Method and system for ultrafast photoelectron microscope

ABSTRACT

An ultrafast system (and methods) for characterizing one or more samples. The system includes a stage assembly, which has a sample to be characterized. The system has a laser source that is capable of emitting an optical pulse of less than 1 ps in duration. The system has a cathode coupled to the laser source. In a specific embodiment, the cathode is capable of emitting an electron pulse less than 1 ps in duration. The system has an electron lens assembly adapted to focus the electron pulse onto the sample disposed on the stage. The system has a detector adapted to capture one or more electrons passing through the sample. The one or more electrons passing through the sample is representative of the structure of the sample. The detector provides a signal (e.g., data signal) associated with the one or more electrons passing through the sample that represents the structure of the sample. The system has a processor coupled to the detector. The processor is adapted to process the data signal associated with the one or more electrons passing through the sample to output information associated with the structure of the sample. The system has an output device coupled to the processor. The output device is adapted to output the information associated with the structure of the sample.

CROSS-REFERENCES TO RELATED APPLICATIONS

This application is a continuation of U.S. patent application Ser. No.11/097,837, entitled “Method and System for Ultrafast PhotoelectronMicroscope,” filed Apr. 1, 2005, which claims the benefit of U.S.Provisional Application Ser. No. 60/559,234, filed Apr. 2, 2004,entitled “Ultrafast Photoelectron Microscope,” and U.S. ProvisionalApplication Ser. No. 60/577,90, filed Jun. 7, 2004, entitled“Diffraction, Crystallography, and Microscopy beyond 3D—StructuralDynamics in Space and Time,” commonly assigned, all of which areincorporated herein by reference.

Work described herein has been supported, in part, by NSF grantCHE-0117850. The United States Government may therefore have certainrights in the invention.

BACKGROUND OF THE INVENTION

The present invention relates generally to imaging of objects. Inparticular, the present invention provides methods and systems forimaging one or more objects using one or more pulses of particlescontaining from about one electron to about 10,000 electrons or morepreferably about 10 electrons to about 100 electrons in a transmissionelectron microscope system. More particularly, the present inventionprovides methods and systems for identifying information about one ormore temporal components associated with one or more spatial features ofcertain objects being imaged. Merely by way of example, the inventionhas been applied to imaging certain chemical, physical, and biologicalobjects. The invention, however, can also be applied to otherapplications such as other areas of biology, chemistry (e.g., organic,physical, biochemistry), medicine (e.g., medical devices, diagnostics,analysis, treatments), physical sciences, electronics, semiconductordevices and materials (e.g., silicon, germanium, Group III/V, GroupII/VI), chemicals (e.g., industrial), petrochemical (e.g., gas, oil),any combination of these, and the like. The invention may be applied toapplications involving processing and/or screening of certain compoundsand/or molecules such as an oligomer, a peptide, a nucleic acid, anoligosaccharide, a phospholipid, a polymer, a protein, or a drugcongener preparation, or any other like species and/or entities.Additionally, the invention may be applied to diffraction,crystallography, spectroscopy, and other areas, and the like. Stillfurther, the invention may be applied to monitoringformation/decomposition of materials, film(s), compounds, and/or otherspecies, depending upon the embodiment.

The twentieth century has been witness to certain major advances in ourability to peer into the microscopic world of molecules, thereby givingus unparalleled insights into their static behavior. For example,electron microscopes, particularly the transmission electron microscope(TEM), provide for direct imaging of macromolecular static structureswith spatial resolution of a few angstroms. These conventional electronmicroscope systems have been employed to image, for example, biologicalmacromolecular crystals. Despite these advances in imaging, the dynamicaspects of structural evolution, insight into which is desirable inunderstanding function, cannot be obtained from currently availablestatic images because of the lack of temporal resolution in conventionalelectron microscopy.

When chemical, and especially biological changes, involve complextransient structures with many possible conformations, one must oftenaddress the nature of the three-dimensional (3D) molecular structures,but at different times during the change. In most biologically importantprocesses, the structural change is reversible, which means that uponinitiation of an event and the associated response, a nondestructivereaction occurs that is repeatable. Unfortunately, limitations existwith conventional microscopic techniques. As an example, conventionalelectron microscopes cannot generally produce images with spatialresolution on the biological length scale (ranging from nanometers tomicrometers) with desirable temporal resolutions. These and otherlimitations of conventional techniques are described throughout thepresent specification and more particularly below.

Thus, there is a need in the art for improved methods imaging at atomicscale resolutions for physical, chemical, biological, and other samples.

SUMMARY OF THE INVENTION

According to the present invention, techniques related to the imaging ofobjects are provided. In particular, the present invention providesmethods and systems for imaging one or more objects using one or morepulses of particles containing from about one electron to about 10,000electrons or more preferably about 10 electrons to about 100 electronsin a transmission electron microscope system. More particularly, thepresent invention provides methods and systems for identifyinginformation about one or more temporal components associated with one ormore spatial features of certain objects being imaged. Merely by way ofexample, the invention has been applied to imaging certain chemical,physical, and biological objects. The invention, however, can also beapplied to other applications such as other areas of biology, chemistry(e.g., organic, physical, biochemistry), medicine (e.g., medicaldevices, diagnostics, analysis, treatments), physical sciences,electronics, semiconductor devices and materials (e.g., silicon,germanium, Group III/V, Group II/VI), chemicals (e.g., industrial),petrochemical (e.g., gas, oil), any combination of these, and the like.The invention may be applied to applications involving processing and/orscreening of certain compounds and/or molecules such as an oligomer, apeptide, a nucleic acid, an oligosaccharide, a phospholipid, a polymer,a protein, or a drug congener preparation, or any other like speciesand/or entities. Additionally, the invention may be applied todiffraction, crystallography, spectroscopy, and other areas, and thelike. Still further, the invention may be applied to monitoringformation/decomposition of materials, film(s), compounds, and/or otherspecies, depending upon the embodiment.

At the subcellular level according to certain embodiments, the inventionmay be applied to the interaction of peptides or chemical compounds(drugs) with the active site of enzymes, or any reversible interactionof a peptide or small molecule with a protein surface. The invention maybe applied to the interaction of nucleic acid binding proteins orenzymes with target nucleic acids, for example, the binding oftranscription factors to specific DNA sequences, or the interaction ofenzymes with DNA, either binding and scanning along the surface of DNAor intercalating into the DNA. The invention may be applied to nucleicacid-nucleic acid interactions, for example, the interaction ofribozymes with RNA according to a specific embodiment.

At the cellular level according to other embodiments, the invention mayalso be applied to the interaction of a compound, protein, virus or cellwith a membrane, whereby the compound, protein, virus or cell “attaches”to the membrane and then passes through it. These and other applicationsare described more specifically below.

In a specific embodiment, the present invention provides a system forimaging one or more samples, e.g., biological, chemical, physical. Thesystem has a stage assembly, which includes a sample to be imaged.Preferably, the system has a laser source, which is capable of emittingan optical pulse of less than 1 picosecond (“ps”) in duration. Thesystem has a cathode coupled to the laser source. In a preferredembodiment, the cathode is capable of emitting an electron pulse lessthan 1 ps in duration although other pulse durations are possible. Anelectron lens assembly is adapted to focus the electron pulse onto thesample disposed on the stage. A detector is adapted to capture one ormore electrons passing through the sample. The one or more electronspassing through the sample is representative of an image of the sample.The detector provides a signal (e.g., data signal) associated with theone or more electrons passing through the sample that represents theimage of the sample. The system has a processor coupled to the detector.In a preferred embodiment, the processor is adapted to process the datasignal associated with the one or more electrons passing through thesample to output information associated with the image represented bythe sample. An output device is coupled to the processor. The outputdevice is adapted to output the information associated with the imagerepresented by the sample.

In an alternative specific embodiment, the present invention provides amethod of operating a transmission electron microscope comprising alaser source, a cathode, and an electron lens assembly. The methodincludes forming a train of optical pulses. Each of the optical pulsesis characterized by a Full Width Half Maximum (“FWHM”) pulse length ofless than 1 ps in duration. The method includes providing a sample forimaging disposed on a stage assembly and generating a train of electronpulses by impinging the train of optical pulses on the cathode. Thetrain of electron pulses is associated with the train of optical pulses.Each of the electron pulses is characterized by a FWHM pulse length lessthan 1 ps in duration. The method includes directing the train ofelectron pulses toward the sample using at least the electron lensassembly. The method captures a portion of the train of electron pulsesusing a sensing device to derive information associated with an image ofthe sample. The method processes the information associated with theimage of the sample.

In yet another alternative specific embodiment, the present inventionprovides a transmission electron microscope (TEM) system capable ofacquiring time resolved images. The system has a laser producing aphoton pulse and a beam splitter adapted to divide the photon pulse intoan electron generation photon pulse and an initiation photon pulse. Acathode is adapted to produce an electron pulse in response toactivation by the electron generation photon pulse. An optical delaystage is adapted to introduce a time delay between the initiation photonpulse and the electron pulse. The system has a sample irradiated by theinitiation photon pulse and the electron pulse. The system has anelectron detector producing an image of the sample in response to theirradiation of the sample by the electron- pulse. Alternatively, theelectron detector produces other information of the sample in responseto the irradiation of the sample by the electron pulse in otherembodiments.

Still further, the present invention provides a method for formingimages from one or more samples using electron beam pulses. The methodincludes providing a feature of a sample to be imaged. The feature has asize of about 100 nanometers and less according to a specificembodiment. The method includes placing the sample onto a stage assemblyand maintaining the sample on the stage assembly in a vacuumenvironment. The method includes directing one or more pulses ofelectrons toward the feature of the sample. Preferably, the one or morepulses of electrons each has about 10 to about 1000 electrons accordingto certain embodiments, although other particle counts of electrons maybe possible. The method captures a portion of the one or more pulses ofelectrons using a sensing device. The portion of the one or more pulsesof the electrons is associated with an image of the feature of thesample. The method includes transferring information associated with theportion of the one or more pulses of electrons associated with the imageof the feature of the sample from the sensing device to a processingdevice. The method outputs a visual image associated with the feature ofthe sample using at least the information associated with the portion ofthe one or more pulses of electrons associated with the image of thefeature of the sample.

In still a further embodiment, the present invention provides a methodof acquiring time-resolved images using an electron microscope. Themethod includes providing a feature of a sample to be imaged. The methodincludes placing the sample onto a stage assembly and maintaining thesample on the stage assembly in a vacuum environment. The methodincludes directing one or more pulses of electrons toward the feature ofthe sample. In a preferred embodiment, the one or more pulses ofelectrons each having about one to about 1000 electrons. The methodincludes capturing a first portion of the one or more pulses ofelectrons using a sensing device during a first portion of time. Theportion of the one or more pulses of the electrons is associated with afirst image of the feature of the sample during the first portion oftime. The method transfers first information associated with the firstportion of the one or more pulses of electrons associated with the firstimage of the feature of the sample during the first portion of time fromthe sensing device to a processing device. The method captures a secondportion of the one or more pulses of electrons using the sensing deviceduring a second portion of time. The portion of the one or more pulsesof electrons is associated with a second image of the feature of thesample during the second portion of time. The method includestransferring second information associated with the second portion ofthe one or more pulses of electrons associated with the second image ofthe feature of the sample during the second portion of time from thesensing device to the processing device. In a preferred embodiment, thefirst image may have characteristics that are different from the secondimage.

In another particular embodiment, the present invention provides asystem for characterizing one or more samples. The system includes astage assembly, which has a sample to be characterized. The system has alaser source that is capable of emitting an optical pulse of less than 1ps in duration. The system has a cathode coupled to the laser source. Ina specific embodiment, the cathode is capable of emitting an electronpulse less than 1 ps in duration. The system has an electron lensassembly adapted to focus the electron pulse onto the sample disposed onthe stage. The system has a detector adapted to capture one or moreelectrons passing through the sample. The one or more electrons passingthrough the sample is representative of the structure of the sample. Thedetector provides a signal (e.g., data signal) associated with the oneor more electrons passing through the sample that represents thestructure of the sample. The system has a processor coupled to thedetector. The processor is adapted to process the data signal associatedwith the one or more electrons passing through the sample to outputinformation associated with the structure of the sample. The system hasan output device coupled to the processor. The output device is adaptedto output the information associated with the structure of the sample.

Still further, the present invention provides a method for capturinginformation from one or more samples using electron beam pulses, whichis derived from respective pulses of electromagnetic radiation, e.g.laser pulse. The method includes providing a feature of a sample to beimaged. The method includes placing the sample onto a stage assembly,which has been maintained a vacuum environment. The method includesdirecting one or more pulses of electrons toward the feature of thesample. In a specific embodiment, the one or more pulses of electronseach has about one to about 1000 electrons or 10 to 100 electronsdepending upon the application. The method includes capturing a portionof the one or more pulses of electrons using a sensing device. Theportion of the one or more pulses of the electrons is associated with acharacterization (e.g., image, diffraction characteristic) of thefeature of the sample. The method includes transferring informationassociated with the portion of the one or more pulses of electronsassociated with the characterization of the feature of the sample fromthe sensing device to a processing device. Optionally, the methodincludes processing the information using the processing device. Themethod outputs at least one or more indications associated with thefeature of the sample using at least the information associated with theportion of the one or more pulses of electrons associated with the imageof the feature of the sample.

In an alternative particular embodiment of the present invention, amethod of acquiring time-resolved images using an electron microscope isprovided. The method includes providing a feature of a sample to beimaged, the feature having a size and placing the sample onto a stageassembly. The method also includes maintaining the sample on the stageassembly in a vacuum environment and directing a first train of electronpulses toward the feature of the sample, the first train of electronpulses having an average temporal pulse width of less than 1 ps.Additionally, the method includes capturing a first portion of the firsttrain of electron pulses using a sensing device during a first portionof time, the portion of the first train of electron pulses beingassociated with a first image of the feature of the sample during thefirst portion of time and transferring first information associated withthe first portion of the first train of electron pulses associated withthe first image of the feature of the sample during the first portion oftime from the sensing device to a processing device. The method furtherincludes directing a second train of electron pulses toward the featureof the sample, the second train of electron pulses having an averagetemporal pulse width of less than 1 ps and capturing a second portion ofthe second train of electron pulses using the sensing device during asecond portion of time, the portion of the second train of electronpulses being associated with a second image of the feature of the sampleduring the second portion of time. Furthermore, the method includestransferring second information associated with the second portion ofthe second train of electron pulses associated with the second image ofthe feature of the sample during the second portion of time from thesensing device to the processing device.

In yet another alternative particular embodiment of the presentinvention, a method of performing time resolved characterization of asample is provided. The method includes providing a photon pulse andproviding a beam splitter to adapted to divide the photon pulse into anelectron generation photon pulse and an initiation photon pulse. In aparticular embodiment of this aspect of the present invention, thephoton pulse is on of a train of ultrafast photon pulses. The methodalso includes providing a cathode adapted to produce an electron pulsein response to activation by the electron generation photon pulse andproviding an optical delay stage adapted to introduce a time delaybetween the initiation photon pulse and the electron pulse. The methodfurther includes providing a sample irradiated by the initiation photonpulse and the electron pulse and providing an electron detectorproducing an image of the sample in response to the irradiation of thesample by the electron pulse.

In yet another alternative embodiment according to the presentinvention, a spectroscopy system is provided. The system includes alaser producing a photon pulse train, a beam splitter adapted to dividethe photon pulse train into an electron generation photon pulse trainand an spectroscopy photon pulse train, and a non-linear optical elementadapted to tune the wavelength of the spectroscopy photon pulse train toan initiation wavelength. The system also includes a cathode adapted toproduce an electron pulse train in response to activation by theelectron generation photon pulse train and an optical delay stageadapted to introduce a time delay between the spectroscopy photon pulsetrain and the electron pulse train. Furthermore, the system includes asample irradiated by the spectroscopy photon pulse train and theelectron pulse train and an electron detector producing an image of thesample in response to the irradiation of the sample by the electronpulse train.

In addition, embodiments of the present invention provide a method fordetermining temporal characteristics of one or more feature of objectsusing an electron microscope assembly. The method includes providing asample including one or more features, the sample being disposed on astage assembly and generating one or more electron pulses, the one ormore electron pulses having a FWHM pulse length of less than 1 ps induration. The method also includes directing the one or more electronpulses toward the one or more features of the sample during a timeperiod associated with a period of detection and capturing a portion ofthe one or more electron pulses using a sensing device to deriveinformation associated with a characteristic of the one or more featuresof the sample. The method further includes processing at least theinformation to identify a temporal characteristic (e.g., a feature thatmay or may not change with respect to time) of the one or more featuresof the sample.

Many benefits are achieved by way of the present invention overconventional techniques. For example, the present technique providestechniques and systems to image materials and biological structures withthe structural dynamics of the sample resolved in both space and timeaccording to a specific embodiment. Additionally, the present inventionprovides for methods and systems that may use conventional electronictechnologies, including computer codes, which are easy to implement, incertain embodiments. The invention also provides a method and systemoperable for capturing images of samples at temperatures compatible forbiological materials in other embodiments. Depending upon theembodiment, certain methods and systems may be applied to diffraction,imaging, crystallography, spectroscopy, and other techniques. In certainembodiments, the present invention provides methods and systems forimaging small features of biological and/or chemical objects in anon-evasive manner, which is often low energy and does not cause thermaldamage to the object, which are often sensitive to excessive thermalenergy. In certain embodiments, the present method and systems includeprocesses that are “adiabatic” in characteristic. Depending upon theembodiment, one or more of these benefits may be achieved. These andother benefits will be described in more throughout the presentspecification and more particularly below.

Various additional objects, features and advantages of the presentinvention can be more fully appreciated with reference to the detaileddescription and accompanying drawings that follow.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1A is a simplified diagram of an ultrafast photoelectron microscopesystem according to an embodiment of the present invention;

FIG. 1B is a simplified perspective diagram of an ultrafastphotoelectron microscope system according to an embodiment of thepresent invention;

FIG. 1C is a simplified diagram of a computer system for controlling theultrafast photoelectron microscope system according to an embodiment ofthe present invention;

FIG. 1D is a simplified block diagram of computer hardware forcontrolling the ultrafast photoelectron microscope system according toan embodiment of the present invention;

FIGS. 2A-2D are a series of images acquired using an embodiment of thepresent invention;

FIG. 3 is an image acquired using electron pulses according to anembodiment of the present invention;

FIGS. 4A-4D are images obtained using a diffraction mode of operationaccording to an embodiment of the present invention;

FIGS. 5A and 5B are images of a biological sample acquired using anembodiment of the present invention;

FIG. 6 is a simplified timing diagram illustrating the use of a methodaccording to the present invention;

FIG. 7 is a simplified flow diagram of an imaging method according to anembodiment of the present invention;

FIG. 8 is a simplified flow diagram of an alternative imaging methodaccording to an embodiment of the present invention;

FIG. 9A is a simplified flow diagram of yet an alternative imagingmethod according to an embodiment of the present invention;

FIG. 9B is a simplified timing diagram illustrating pulse trainsaccording to an embodiment of the present invention;

FIG. 9C is a simplified flowchart illustrating image collectionaccording to one embodiment of the present invention;

FIG. 10 is a simplified flow diagram of a still an alternative imagingmethod according to an embodiment of the present invention;

FIGS. 11 through 15 are simplified diagrams of experimental resultsaccording to embodiments of the present invention;

FIG. 16 is a simplified diagram of an experimental method according toan alternative embodiment of the present invention;

FIGS. 17 through 19 are simplified diagrams of experimental resultsaccording to alternative embodiments of the present invention; and

FIG. 20 is a simplified diagram of experimental results according to aspecific embodiment of the present invention.

DETAILED DESCRIPTION OF SPECIFIC EMBODIMENTS

According to the present invention, techniques related to the imaging ofobjects are provided. In particular, the present invention providesmethods and systems for imaging one or more objects using one or morepulses of particles containing from about one electron to about 10,000electrons or more preferably about 10 electrons to about 100 electronsin a transmission electron microscope system. More particularly, thepresent invention provides methods and systems for identifyinginformation about one or more temporal components associated with one ormore spatial features of certain objects being imaged. Merely by way ofexample, the invention has been applied to imaging certain chemical,physical, and biological objects. The invention, however, can also beapplied to other applications such as other areas of biology, chemistry(e.g., organic, physical, biochemistry), medicine (e.g., medicaldevices, diagnostics, analysis, treatments), physical sciences,electronics, semiconductor devices and materials (e.g., silicon,germanium, Group III/V, Group II/VI), chemicals (e.g., industrial),petrochemical (e.g., gas, oil), any combination of these, and the like.The invention may be applied to applications involving processing and/orscreening of certain compounds and/or molecules such as an oligomer, apeptide, a nucleic acid, an oligosaccharide, a phospholipid, a polymer,a protein, or a drug congener preparation, or any other like speciesand/or entities. Additionally, the invention may be applied todiffraction, crystallography, spectroscopy, and other areas, and thelike. Still further, the invention may be applied to monitoringformation/decomposition of materials, film(s), compounds, and/or otherspecies, depending upon the embodiment. Details of embodiments of thepresent invention can be found throughout the specification and moreparticularly below. Before discussing details of the variousembodiments, we have provided certain information, which we have learnedand/or discovered, as may be applied to a description of suchembodiments below.

Beginning with X-rays at the turn of the 20th century, diffractiontechniques have allowed determination of equilibrium three-dimensionalstructures with atomic resolution, in systems ranging from diatoms(NaCl) to DNA, proteins, and complex assemblies such as viruses. Fordynamics, the time resolution has similarly reached the fundamentalatomic-scale of motion. With the advent of femtosecond time resolutionnearly two decades ago, it has become possible to study the dynamics ofnon-equilibrium molecular systems in real time: from the very small(NaI) to the very large (DNA, proteins and their complexes) as will beexplained below.

Armed with this ability to capture both the static architecture as wellas the temporal behavior of the chemical bond, a desire that nowstimulates researchers the world over is the potential to map out, inreal time, the coordinates of all individual atoms in a reaction, as,for example, when a molecule unfolds to form selective conformations, orwhen a protein docks onto the cell surface. These transient structuresprovide important insights into the function of chemical and biologicalmolecules. As function is intimately associated with intrinsicconformational dynamics, knowing a molecule's static structure is oftenonly the first step toward unraveling how the molecule functions,especially in the world of biology. Thus, elucidating the real-time“structural dynamics” of far-from-equilibrium conformations at atomicscale resolution is vital to understanding the fundamental mechanisms ofcomplex chemical and biological systems.

Time-resolved experiments with femtosecond time resolution have beenperformed in the past with probe wavelengths ranging from theultraviolet to the infrared and far infrared. On this time scale, one isable to freeze localized structures in space (wave packets) and observetheir evolution in time—thus elucidating the elementary processes ofbond transformation via transition states, in chemistry and biology.

Certain advances have been made in multidimensional spectroscopy tocorrelate frequencies of optical transitions with temporal evolution,thereby probing structural changes in different relaxation processes.For complex molecular structures, however, the positions of all atoms ata given time can only be obtained if the probe is able to “see”interferences of all atoms. Diffraction methods using X-rays orelectrons have the unique ability of revealing all internuclearcoordinates with very high spatial resolution, thus providing a globalpicture of structural change on the ultrafast time scale with atomiclevel detail.

Diffraction techniques using electron or X-ray pulses can, in principle,be used to obtain certain time-varying molecular structures. Thesepulses often must be short enough to freeze the atomic motions, yetbright enough to provide a discernible diffraction pattern. In the caseof X-rays, photons are scattered by electrons in the molecular sample,so the diffracted intensity depends directly on the electronic density.Because most electrons are centered on atoms, these electron densitiesreflect the positions of nuclei, especially for heavy atoms. Ultrafastpulsed X-ray sources include third-generation synchrotron radiation,laser produced plasma sources, high-order harmonics production in gasesand on solid surfaces, and free-electron lasers. While high-flux X-raypulses from synchrotron sources are relatively long (tens ofpicoseconds; dictated by the duration of electron bunches in a storagering), the sub-picosecond X-ray pulses from other generation schemessuffer from rather low fluxes. As a result, ultrafast X-ray diffractionstudies have primarily focused on solid samples where the intrinsiclong-range order enhances the signal-to-noise ratio of the interferencepatterns. X-Ray absorption spectroscopy (XAS) techniques such asextended X-ray absorption fine structure (EXAFS) and X-ray absorptionnear-edge structure (XANES) spectroscopy have been used to obtain localstructural information in solutions on the nanosecond timescale, and onthe ultrafast timescale, in gases and liquids.

Electron diffraction provides numerous benefits. For example, thecross-section for electron scattering is about six orders of magnitudelarger than that for X-ray scattering. Moreover, the experiments are ofa “tabletop” scale and can be implemented with ultrafast (femtosecondand picosecond) laser sources. Electrons produce less damage tospecimens per useful elastic scattering event than X-rays. Electrons,because of their short penetration depth arising from their stronginteraction with matter, can reveal transient structures of gases,surfaces, and (thin) crystals. Electrons can be focused to obtain imagesin microscopy. As described more fully below, using properly timedsequences of electron pulses, sometimes referred to as framereferencing, one is able to “isolate” changing transient structures.Depending upon the embodiment, there can also be other benefits.

Embodiments of the present invention provide methods and systems totemporally and spatially resolved transient structures includingstructures in radiationless transitions, structures in non-concertedorganic reactions, structures in non-concerted organometallic reactions,structures of carbene intermediates, dynamic pseudorotary structures,non-equilibrium structures and conformational structures on complexenergy landscapes, and hydrogen-bonded structures of tautomericreactions. Merely by way of example, the reaction of pyridine, which wasbelieved to form valence structures was shown to have a reaction pathwaythat involves the opening of the ring with the formation of a diradicalintermediate.

Optical microscopy, using fluorescent probes e.g., green fluorescentproteins, has provided the means to visualize events occurring in vitroand within cells. But although optical methods, in principle, canprovide temporal resolution on the scale of femtoseconds, they arelimited in the spatial resolution achievable to resolution on the orderof the wavelength of the light used in the microscope, typically 200 nm.Other limitations may also exist using optical microscopy.

Direct imaging of macromolecular static structures with resolution of afew angstroms has been employed with great success in conventionalelectron microscopy of many systems, including biological macromolecularcrystals. However, dynamics of structural change, which are critical toour understanding of the function, cannot be obtained without timeresolution. The development of a stroboscopic methodology allows for theinvestigation of structural dynamics with a time-scale limited only bythe temporal characteristics of the electron probe. This separation oftime-scales limits energy dissipation, which at longer times usuallyleads to structural disintegration. As importantly, on thesub-picosecond time-scale, the motion of the atoms is negligible. Insome embodiments of the present invention, the spatial structure inthree dimensions (3D) is imaged as a function of time (4D) to provideinsight, for example, into structural dynamics.

Using embodiments of the present invention, samples are preparedcoherently, resolving issues related to the time-energy uncertaintyprinciple in limiting the information to be gained fromfemtosecond-to-picosecond time resolution. Moreover, for any dynamicprocess, changes as a function of time are continuous, and although someglobal events may occur at longer times, sometimes referred to as“relevant time scales,” these events are triggered by changes at earlytimes. The primary events are an essential part of any completedescription of the landscape and the dynamics. Thus the notion that“relevant” biological events occur only far beyond the ultrafast timedomain gives an incomplete picture and may prove, as in early notions ofchemical reactions to be misleading.

In certain embodiments of the present invention, the term “ultrafast” isused to characterize various components of systems described herein. Oneof skill in the art appreciates that the term ultrafast refers topulses, containing either photons or electrons, shorter than 1picosecond (“ps”) in duration. The measurement of the pulse width isgenerally performed at the full width half maximum (FWHM) of the pulse,although this is not required by the present invention. Generally,pulses with widths on the order of 100 fs are utilized in embodiments ofthe present invention, well under the threshold of 1 ps defining theterm ultrafast. Of course, there can also be other variations,modifications, and alternatives. Certain details of present methods andsystems can be found through out the present specification and moreparticularly below.

FIG. 1A is a simplified diagram of an ultrafast photoelectron microscopesystem according to an embodiment of the present invention. This diagramis merely an example, which should not unduly limit the scope of theclaims herein. One of ordinary skill in the art would recognize manyvariations, modifications, and alternatives. As illustrated in FIG. 1A,a femtosecond laser 110 is directed through a Pockels cell 112, whichacts as a controllable shutter. A Glan polarizer 114 is used in someembodiments, to select the laser power propagating in optical path 115.A beam splitter (not shown) is used to provide several laser beams tovarious portions of the system. Although the system illustrated in FIG.1A is described with respect to imaging applications, this is notgenerally required by the present invention. One of skill in the artwill appreciate that embodiments of the present invention providesystems and methods for imaging, diffraction, crystallography, andrelated fields. Particularly, the experimental results discussed belowyield insight into the varied applications available using embodimentsof the present invention.

The femtosecond laser source 110 is generally capable of generating atrain of optical pulses with predetermined pulse width. One example ofsuch a laser system is a diode-pumped mode-locked titanium sapphire(Ti:Sapphire) laser oscillator operating at 800 nm and generating 100 fspulses at a repetition rate of 80 MHz and an average power of 1 Watt,resulting in a period between pulses of 12.5 ns. In an embodiment, thespectral bandwidth of the laser pulses is 2.35 nm FWHM. An example ofone such laser is a Mai Tai One Box Femtosecond Ti:Sapphire Laser,available from Spectra-Physics Lasers, of Mountain View, Calif. Inalternative embodiments, other laser sources generating optical pulsesat different wavelengths, with different pulse widths, and at differentrepetition rates are utilized. One of ordinary skill in the art wouldrecognize many variations, modifications, and alternatives.

A first portion of the output of the femtosecond laser 110 is coupled toa second harmonic generation (SHG) device 116, for example a bariumborate (BaB₂O₄) crystal, typically referred to as a BBO crystal andavailable from a variety of doubling crystal manufacturers. The SHGdevice frequency doubles the train of optical pulses to generate a trainof 400 nm, 100 fs optical pulses at an 80 MHz repetition rate. SHGdevices generally utilize a nonlinear crystal to frequency double theinput pulse while preserving the pulse width. In some embodiments, theSHG is a frequency tripling device, thereby generating an optical pulseat UV wavelengths. Of course, the desired output wavelength for theoptical pulse will depend on the particular application. The doubledoptical pulse produced by the SHG device propagates along electrongenerating path 118.

A cw diode laser 120 is combined with the frequency doubled opticalpulse using beam splitter 122. The light produce by the cw diode laser,now collinear with the optical pulse produced by the SHG device, servesas an alignment marker beam and is used to track the position of theoptical pulse train in the electron generating path. The collinear laserbeams enter chamber 130 through entrance window 132. In the embodimentillustrated in FIG. 1A, the entrance window is fabricated from materialswith high transparency at 400 nm and sufficient thickness to providemechanical rigidity. For example, BK-7 glass about 6 mm thick withanti-reflection coatings, e.g. MgF₂ or sapphire are used in variousembodiments. One of ordinary skill in the art would recognize manyvariations, modifications, and alternatives.

An optical system, partly provided outside chamber 130 and partlyprovided inside chamber 130 is used to direct the frequency doubledoptical pulse train along the electron-generating path 134 inside thechamber 130 so that the optical pulses impinge on cathode 140. Asillustrated, the optical system includes mirror 144, which serves as aturning mirror inside chamber 130. In embodiments of the presentinvention, polished metal mirrors are utilized inside the chamber 130since electron irradiation may damage mirror coatings used on someoptical mirrors. In a specific embodiment, mirror 144 is fabricated froman aluminum substrate that is diamond turned to produce a mirrorsurface. In some embodiments, the aluminum mirror is not coated. Inother embodiments, other metal mirrors, such as a mirror fabricated fromplatinum is used as mirror 144.

In an embodiment, the area of interaction on the cathode was selected tobe a flat 300 μm in diameter. Moreover, in the embodiment illustrated,the frequency doubled optical pulse was shaped to provide a beam with abeam waist of a predetermined diameter at the surface of the cathode. Ina specific embodiment, the beam waist was about 50 μm. In alternativeembodiments, the beam waist ranged from about 30 μm to about 200 μm. Ofcourse, the particular dimensions will depend on the particularapplications. The frequency doubled optical pulse train was steeredinside the chamber using a computer controlled mirror in a specificembodiment.

In a specific embodiment, the optical pulse train is directed toward afront-illuminated photocathode where the irradiation of the cathode bythe laser results in the generation of electron pulses via thephotoelectric effect. Irradiation of a cathode with light having anenergy above the work function of the cathode leads to the ejection ofphotoelectrons. That is, a pulse of electromagnetic energy above thework function of the cathode ejects a pulse of electrons according to apreferred embodiment. Generally, the cathode is maintained at atemperature of 1000 K, well below the thermal emission thresholdtemperature of about 1500 K, but this is not required by the presentinvention. In alternative embodiments, the cathode is maintained at roomtemperature. In some embodiments, the cathode is adapted to provide anelectron pulse of predetermined pulse width. The trajectory of theelectrons after emission follows the lens design of the TEM, namely thecondenser, the objective, and the projector lenses. Depending upon theembodiment, there may also be other configurations.

In the embodiment illustrated, the cathode is a Mini-Vogel mount singlecrystal lanthanum hexaboride (LaB₆) cathode shaped as a truncated conewith a flat of 300 μm at the apex and a cone angle of 90°, availablefrom Applied Physics Technologies, Inc., of McMinnville, Oreg. As isoften known, LaB₆ cathodes are regularly used in transmission andscanning electron microscopes. The quantum efficiency of LaB₆ cathodesis about 10⁻³ and these cathodes are capable of producing electronpulses with temporal pulse widths on the order of 10⁻¹³ seconds. In someembodiments, the brightness of electron pulses produced by the cathodeis on the order of 10⁹ A/cm²/rad² and the energy spread of the electronpulses is on the order of 0.1 eV. In other embodiments, the pulse energyof the laser pulse is reduced to about 500 pJ per pulse, resulting inapproximately one electron/pulse

Generally, the image quality acquired using a TEM is proportional to thenumber of electrons passing through the sample. That is, as the numberof electrons passing through the sample is increased, the image qualityincreases. Some pulsed lasers, such as some Q-switched lasers, reducethe pulse count to produce a smaller number of pulses characterized byhigher peak power per pulse. Thus, some laser amplifiers operate at a 1kHz repetition rate, producing pulses with energies ranging from about 1μJ to about 2 mJ per pulse. However, when such high peak power lasersare used to generate electron pulses using the photoelectric effect,among other issues, both spatial and temporal broadening of the electronpulses adversely impact the pulse width of the electron pulse or packetproduced. In some embodiments of the present invention, the laser isoperated to produce low power pulses at higher repetition rates, forexample, 80 MHz. In this mode of operation, benefits available usinglower power per pulse are provided, as described below. Additionally,because of the high repetition rate, sufficient numbers of electrons areavailable to acquire high quality images.

In some embodiments of the present invention, the laser power ismaintained at a level of less than 500 pJ per pulse to prevent damage tothe photocathode. As a benefit, the robustness of the photoemitter isenhanced. Additionally, laser pulses at these power levels preventspace-charge broadening of the electron pulse width during the flighttime from the cathode to the sample, thus preserving the desiredfemtosecond temporal resolution. Additionally, the low electron countper pulse provided by some embodiments of the present invention reducesthe effects of space charge repulsion in the electron pulse, therebyenhancing the focusing properties of the system. As one of skill in theart will appreciated, a low electron count per pulse, coupled with ahigh repetition rate of up to 80 MHz provided by the femtosecond laser,provides a total dose as high as one electron/A as generally utilized inimaging applications.

In alternative embodiments, other suitable cathodes capable of providinga ultrafast pulse of electrons in response to an ultrafast optical pulseof appropriate wavelength are utilized. In embodiments of the presentinvention, the cathode is selected to provide a work function correlatedwith the wavelength of the optical pulses provided by the SHG device.The wavelength of radiation is related to the energy of the photon bythe familiar relation λ(μm)˜1.24÷v (eV), where λ is the wavelength inmicrons and v is the energy in eV. For example, a LaB₆ cathode with awork function of 2.7 eV is matched to optical pulses with a wavelengthof 400 nm (v=3.1 eV) in an embodiment of the present invention. Asillustrated, the cathode is enclosed in a vacuum chamber 130, forexample, a housing for a transmission electron microscope (TEM). Ingeneral, the vacuum in the chamber 130 is maintained at a level of lessthan 1×10⁻⁶ torr. In alternative embodiments, the vacuum level variesfrom about 1×10⁻⁶ torr to about 1×10⁻¹⁰ torr. The particular vacuumlevel will be a function of the varied applications.

In embodiments of the present invention, the short duration of thephoton pulse leads to ejection of photoelectrons before an appreciableamount of the deposited energy is transferred to the lattice of thecathode. In general, the characteristic time for thermalization of thedeposited energy in metals is below a few picoseconds, thus no heatingof the cathode takes place using embodiments of the present invention.

Electrons produced by the cathode 140 are accelerated past the anode 142and are collimated and focused by electron lens assembly 146 anddirected along electron imaging path 148 toward the sample 150. Theelectron lens assembly generally contains a number of electromagneticlenses, apertures, and other elements as will be appreciated by one ofskill in the art. Electron lens assemblies suitable for embodiments ofthe present invention are often used in TEMs. The electron pulsepropagating along electron imaging path 148 is controlled in embodimentsof the present invention by a controller (not shown, but described inmore detail with reference to certain Figures below) to provide anelectron beam of predetermined dimensions, the electron beam comprisinga train of ultrafast electron pulses.

The relationship between the electron wavelength (λ_(deBroglie)) and theaccelerating voltage (U) in an electron microscope is given by therelationship λ_(deBroglie)=h/(2m₀eU)^(1/2), where h, m₀, e are Planck'sconstant, the electron mass, and an elementary charge. As an example,the de Broglie wavelength of an electron pulse at 120 kV corresponds to0.0335 Å, and can be varied depending on the particular application. Thebandwidth or energy spread of an electron packet is a function of thephotoelectric process and bandwidth of the optical pulse used togenerate the electron packet or pulse.

Electrons passing through the sample or specimen 150 are focused byelectron lens assembly 152 onto a detector 154. Although FIG. 1Aillustrates two electron lens assemblies 146 and 152, the presentinvention is not limited to this arrangement and can have other lensassemblies or lens assembly configurations. In alternative embodiments,additional electromagnets, apertures, other elements, and the like areutilized to focus the electron beam either prior to or after interactionwith the sample, or both.

Detection of electrons passing through the sample, includingsingle-electron detection, is achieved in one particular embodimentthrough the use of an ultrahigh sensitivity (UHS) phosphor scintillatordetector 154 especially suitable for low-dose applications inconjunction with a digital CCD camera. In a specific embodiment, the CCDcamera was an UltraScan™ 1000 UHS camera, manufactured by Gatan, Inc.,of Pleasanton, Calif. The UltraScan™ 1000 CCD camera is a 4 mega-pixel(2048×2048) camera with a pixel size of 14 μm×14 μm, 16-bitdigitization, and a readout speed of 4 Mpixels/sec. In the embodimentillustrated, the digital CCD camera is mounted under the microscope inan on-axis, below the chamber position. In order to reduce the noise andpicture artifacts, in some embodiments, the CCD camera chip isthermoelectrically cooled using a Peltier cooler to a temperature ofabout −25° C. The images from the CCD camera were obtained withDigitalMicrograph™ software embedded in the Tecnai™ user interface, alsoavailable from Gatan, Inc. Of course, there can be other variations tothe CCD camera, cooler, and computer software, depending upon theembodiment.

FIG. 1B illustrates an embodiment of the present invention in which aTEM is modified to provide a train of electron pulses used for imagingin addition to the thermionic emission source used for imaging ofsamples. Merely by way of example, an FEI Tecnai™ G² 12 TWIN, availablefrom FEI Company in Hillsboro, Oreg., may be modified according toembodiments of the present invention. The Tecnai™ G² 12 TWIN is anall-in-one 120 kV (λ_(deBroglie)=0.0335 Å) high-resolution TEM optimizedfor 2D and 3D imaging at both room ad liquid-nitrogen temperatures.Embodiments of the present invention leverage capabilities provided bycommercial TEMs such as automation software, detectors, data transfertechnology, and tomography.

In particular, in some embodiments of the present invention, afive-axis, motor-driven, precision goniometer is used with computersoftware to provide automated specimen tilt combined with automatedacquisition of images as part of a computerized tomography (CT) imagingsystem. In these embodiments, a series of 2D images are captured atvarious specimen positions and combined using computer software togenerate a reconstructed 3D image of the specimen. In some embodiments,the CT software is integrated with other TEM software and in otherembodiments, the CT software is provided off-line. One of ordinary skillin the art would recognize many variations, modifications, andalternatives.

In certain embodiments in which low-electron content electron pulses areused to image the sample, the radiation damage is limited to the transitof the electrons in the electron pulses through the sample. Typically,samples are on the order of 100 nm thick, although other thicknesseswould work as long as certain electrons may traverse through the sample.Thus, the impact of radiation damage on these low-electron contentelectron pulse images is limited to the damage occurring during thistransit time. Radiation induced structural damage occurring on longertime scales than the transit time will not impact the collected image,as these damage events will occur after the structural information iscollected.

Utilizing the apparatus described thus far, embodiments of the presentinvention provide systems and methods for imaging material andbiological specimens both spatially and temporally with atomic-scalespatial resolution on the order of 1 nm and temporal resolution on theorder of 100 fs. At these time scales, energy randomization is limitedand the atoms are nearly frozen in place,.thus methods according to thepresent invention open the door to time-resolved studies of structuraldynamics at the atomic scale in both space and time. Details of thepresent computer system according to an embodiment of the presentinvention may be explained according to the description below.

FIG. 1C is a simplified diagram of a computer system 210 that is used tooversee the system of FIG. 1A and 1B according to an embodiment of thepresent invention. This diagram is merely an example, which should notunduly limit the scope of the claims herein. One of ordinary skill inthe art would recognize many other modifications, alternatives, andvariations. As shown, the computer system 210 includes display device220, display screen 230, cabinet 240, keyboard 250, and mouse 270. Mouse270 and keyboard 250 are representative “user input devices.” Mouse 270includes buttons 280 for selection of buttons on a graphical userinterface device. Other examples of user input devices are a touchscreen, light pen, track ball, data glove, microphone, and so forth.

The system is merely representative of but one type of system forembodying the present invention. It will be readily apparent to one ofordinary skill in the art that many system types and configurations aresuitable for use in conjunction with the present invention. In apreferred embodiment, computer system 210 includes a Pentium™ classbased computer, running Windows™ NT or XP operating system by MicrosoftCorporation. However, the system is easily adapted to other operatingsystems such as any open source system and architectures by those ofordinary skill in the art without departing from the scope of thepresent invention. As noted, mouse 270 can have one or more buttons suchas buttons 280. Cabinet 240 houses familiar computer components such asdisk drives, a processor, storage device, etc. Storage devices include,but are not limited to, disk drives, magnetic tape, solid-state memory,bubble memory, etc. Cabinet 240 can include additional hardware such asinput/output (I/O) interface cards for connecting computer system 210 toexternal devices external storage, other computers or additionalperipherals, which are further described below.

FIG. 1D is a more detailed diagram of hardware elements in the computersystem of FIG. 1C according to an embodiment of the present invention.This diagram is merely an example, which should not unduly limit thescope of the claims herein. One of ordinary skill in the art wouldrecognize many other modifications, alternatives, and variations. Asshown, basic subsystems are included in computer system 210. In specificembodiments, the subsystems are interconnected via a system bus 275.Additional subsystems such as a printer 274, keyboard 278, fixed disk279, monitor 276, which is coupled to display adapter 282, and othersare shown. Peripherals and input/output (I/O) devices, which couple toI/O controller 271, can be connected to the computer system by anynumber of means known in the art, such as serial port 277. For example,serial port 277 can be used to connect the computer system to a modem281, which in turn connects to a wide area network such as the Internet,a mouse input device, or a scanner. The interconnection via system busallows central processor 273 to communicate with each subsystem and tocontrol the execution of instructions from system memory 272 or thefixed disk 279, as well as the exchange of information betweensubsystems. Other arrangements of subsystems and interconnections arereadily achievable by those of ordinary skill in the art. System memory,and the fixed disk are examples of tangible media for storage ofcomputer programs, other types of tangible media include floppy disks,removable hard disks, optical storage media such as CD-ROMS and barcodes, and semiconductor memories such as flash memory,read-only-memories (ROM), and battery backed memory.

Although the above has been illustrated in terms of specific hardwarefeatures, it would be recognized that many variations, alternatives, andmodifications can exist. For example, any of the hardware features canbe further combined, or even separated. The features can also beimplemented, in part, through software or a combination of hardware andsoftware. The hardware and software can be further integrated or lessintegrated depending upon the application. Further details of thefunctionality, which may be carried out using a combination of hardwareand/or software elements, of the present invention can be outlined belowaccording to the Figures.

FIGS. 2A-2D are a series of images acquired using an embodiment of thepresent invention. In order to characterize system performance, acalibration specimen was placed in the system described above and imagedusing multiple methodologies. In the embodiment illustrated in FIG. 2,the specimen was a replica of a 2,160 lines/mm waffle-patterndiffraction grating. The spacing between lines for the specimen was0.462 μm and the waffle-pattern is first visible around a magnificationof ×2500.

FIG. 2A is an image acquired using electrons emitted via thermionicemission from an electron gun. As is known to one of skill in the art,conventional electron guns used in TEMs utilize thermionic emission togenerate a stream of electrons that are accelerated away from thecathode and focused into a beam directed toward the sample. In FIG. 2A,the voltage between the cathode and the anode was 120 kV and themagnification of the imaging system was ×4400. The scale marker 290denotes 0.5 μm, which is approximately the spacing between adjacentlines of 0.463 μm.

FIG. 2B is an image acquired using electrons emitted via thephotoelectric effect from the same cathode used to create the image inFIG. 2A. As described above, using embodiments of the present invention,a train of photon pulses is directed onto the cathode, generating atrain of electron pulses that travel along the imaging path. Operatingthe microscope at the same 120 kV voltage, the stream of electron pulsesencounter the specimen with a portion of the electron pulses passingthrough the sample, resulting in the image shown in FIG. 2B. Therefore,utilizing methods and systems according to embodiments of the presentinvention, it is possible to acquire images of static specimens.

For calibration purposes, background “images” were obtained with thecathode turned off (i.e. no thermionic emission) and the femtosecondpulses blocked prior to entering the chamber 130. These “images” were ofan opaque background scene comprising readout noise, demonstrating thatthe image illustrated in FIG. 2B resulted from the photoelectric effectand the production of electrons by thermal emission processes wasnegligible. Although it is possible to generate thermal electrons bylaser heating of the cathode and/or as the result of the resistanceheating of the cathode, the calibration measurements eliminated thesepossibilities.

FIGS. 2C and 2D are images acquired using a thermionic emission sourceand electron pulses, respectively. The scale marker 292 denotes adistance of 100 nm, approximately a fifth of the spacing betweenadjacent lines. Shifting of the specimen between measurement is observedin the images.

FIG. 3 is an image acquired using electron pulses according to anembodiment of the present invention. The object shown is graphitizedcarbon on porous carbon grids. The magnification for the image shown is×1110000 and the scale bar 310 is 20 nm. A calibration “image” wasobtained with the femtosecond pulses blocked prior to entering thechamber, resulting in an opaque background scene comprising readoutnoise.

FIGS. 4A-4C are images obtained using a diffraction mode of operationaccording to an embodiment of the present invention. In order to acquirethe image shown in the Figures, the intermediate lens (not shown in FIG.1A) was adjusted to select the back focal plane of the objective lens asits object. The diffraction patterns were collected at 120 kV in boththermionic emission and electron pulse mode for samples ofpolycrystalline aluminum and single-crystal gold. FIG. 4A is adiffraction pattern with a scale 410 of 5 nm⁻¹ acquired using thermionicemission from a sample of polycrystalline aluminum. FIG. 4B is adiffraction pattern with the same scale acquired using a train ofultrafast electron pulses on the same polycrystalline aluminum sample.Similar diffraction patterns were collected using thermionic emission(FIG. 4C) and a train of ultrafast electron pulses (FIG. 4D) using asingle crystal gold sample. The diffraction patterns illustrated may beindexed to provide the atomic plane spacings and symmetry depending uponthe embodiment.

One of skill in the art will appreciate that embodiments of the presentinvention are not limited to imaging applications, but may also includediffraction and crystallography applications. As will be described withreference to certain Figures below, optically initiated diffractionexperiments are provided by embodiments of the present invention, butare not limited to this particular application. Thus, it will beappreciated that embodiments of the present invention encompass imagingand diffraction experiments using electron pulses. Moreover, imaging anddiffraction experiments in which a sample is optically initiated orspectroscopically activated are also encompassed by embodiments of thepresent invention. Of course, there can be other variations,modifications, and alternatives.

FIG. 5 is an image of a biological sample acquired using an embodimentof the present invention. In FIG. 5, a positively-stained (uranylacetate) biological cell, embedded in a resin, in particular, a ratintestine, was imaged using the system illustrated in FIG. 1A. Althoughsample preparation for this embodiment was performed using aconventional positive staining protocol, this is not required by thepresent invention. FIG. 5A was acquired using an electron beam generatedusing thermionic emission. FIG. 5B was acquired using a train ofultrafast electron pulses. The image shown in FIG. 5B, acquired in amatter of several seconds, using femtosecond electron pulses with alow-electron content of approximately one electron per pulse, providestime resolved images unavailable using thermionic emission sources.

The ultrafast time resolution provided by an ultrafast electronmicroscope (UEM) according to embodiments of the present invention hasunique consequences. For the image shown in FIG. 5B, energyrandomization is limited as the atoms are nearly frozen in place by thestroboscopic method provided by embodiments of the present invention.Thus, studies of intact biological samples are enabled by embodiments ofthe present invention. Using the methods and systems described herein,it is possible to extend the results achieved using cryo-electronmicroscopes (characterized by resolution on the order of milliseconds asdictated by freezing/mixing rates) to study the structure, assembly anddynamics of macromolecules with time resolutions in the ultrafastregime. The low number of electrons in each ultrafast pulse, along withestablished methods of cryofixation, present a tremendous advance inlimiting damage from electron irradiation as discussed previously.

It should be noted that for sample in which biological structuralrecovery is longer than 12.5 ns, pulse pickers, such as the Pulse-Picker9200, available from Coherent, Inc., of Santa Clara, Calif., may be usedto select pulses from the train at rates suitable for such a recovery.Moreover, as illustrated in FIGS. 2, 4A-D, and 5, embodiments of thepresent invention provide methods and systems for alternativelyobtaining either images or diffraction patterns using certainconventional TEM modes of operation or in the UEM mode. Thus, withminimal system modifications and training, embodiments of the presentinvention provide numerous benefits.

Referring once again to the optical path 115 in FIG. 1A, a secondportion of the optical pulse produced by laser 110 is coupled to theoptical parametric oscillator (OPO) 160. The OPO 160 provides tunablefemtosecond pulses in the infrared from about 1.1 μm to about 2.25 μm byparametric generation of two longer wavelength photons from a singleshorter wavelength photon. In addition, doubling of the frequency ofinfrared pulses produced by the OPO further extends access to a visibleregion of a spectrum. In some embodiments, the OPO enhances theversatility of the ultrafast Ti:Sapphire laser by extending its outputinto the visible and infrared regions of the spectrum. In someembodiments of the present invention, the OPO 160 is utilized to tunethe photon beam for spectroscopic applications. For example, in somespectroscopy applications, the frequency associated with a pump beamused to initiate, for example, a chemical reaction in a sample, isselected to maximize the interaction between the pump beam and thesample. Thus, in this particular application, the OPO is utilized totune the frequency of the photon beam in path 164 as desired. Thespectroscopic applications available through embodiments of the presentinvention are not limited to chemical reactions, but may includeabsorption spectroscopy, electron energy loss spectroscopy (EELS), andother applications.

An optical delay stage 162 is provided in change-initiating path 164 tointroduce a predetermined delay in the optical path of the opticalpulses propagating along path 164. One of skill in the art willappreciate techniques for introducing and controlling an optical delaystage such as that illustrated in FIG. 1A. In some embodiments, theoptical delay stage 162 is used to determine a “zero time” in which anoptical pulse in the change-initiating path 164 and an electron pulse inthe electron imaging path 148 are temporally and spatially aligned toarrive at the sample 150 simultaneously. In other embodiments, theoptical delay is selectable to introduce either positive (optical pulsedelayed a selected time period with respect to the electron pulse) ornegative (optical pulse advanced a selected time period with respect tothe electron pulse). One of ordinary skill in the art would recognizemany variations, modifications, and alternatives. The optically delayedpulse enters chamber 130 through window 166 and is reflected off mirror168 and directed toward the sample 150. In some embodiments, an imagingsystem 170 is provided including coaxial illuminator 172 and CCD camera174 to provide for monitoring of the sample using light scattered backoff the surface of the sample 150. As will be appreciated by one ofskill in the art, many variations, modifications, and alternatives areavailable for the imaging system 170. Window 166 and mirror 168 areidentical to window 132 and mirror 144 in some embodiments, althoughthis is not required by the present invention.

Although the previous discussion describes an optical delay stage in theoptical path 164, this is not required by the present invention.Depending upon the embodiment, an optical delay stage could beintegrated into electron generating path 118 to delay the generation ofthe electron beam with respect to the optical beam in change-inducingpath 164. As mentioned previously, the delay in the electron generatingpath may be positive or negative as the stage is generally configuredwith a negative delay at one extreme, a positive delay at the otherextreme, and a zero-time delay at an intermediate portion of the stage.

In embodiments of the present invention utilizing the optical pulse inthe change-inducing path 164 to initiate a change in the sample, thetime coordinate for a reaction is generally established based on a pointof reference for the relative time delay between the optical initiationpulse and the electron pulse. This reference point is commonly referredto as time-zero (t₀), the time when both pulses simultaneously intersectin the sample. One approach to determining time-zero is based on acareful measurement of photon and electron beam paths. This techniquecan typically narrow the time-zero window to within 100 ps.

Another approach utilized in some embodiments of the present inventionis to use the crossed-beam geometry of an actual diffraction experimentto determine time-zero via the “lensing effect.” For example, duringCF₃I dissociation reaction studies, we observed a dramatic change in theundiffracted electron beam profile when the excitation laser waspresent. The beam spot intensified along one axis, with a correspondingsubtle decrease in the overall width. This effect only occurred whenboth the excitation laser and the molecular beam were present. Theintensified strip was parallel to the laser axis and could be shifted upand down within the beam spot by adjusting the vertical tilt of theexcitation laser entrance lens. Defocusing the laser reduced the stripeintensity. We refer to this phenomenon photoionization-induced lensing.The effect is analogous to plasma lensing, a technique in which thehigh-energy charged beams in particle accelerators are focused bypassing through a plasma field.

In yet another embodiment of the present invention, an in-situsynchronization method is utilized with an energy filter tuned in aregion corresponding to resonant plasmon loss. Because theelectron-electron scattering constant is on the order of a fewfemtoseconds, the position of the plasmon peak is a natural marker forexperimental synchronization. In particular, the position of the peak inenergy loss spectrum corresponds to the plasmon frequency and isproportional to the free carriers in the sample. The plasmon resonanceis the strongest feature in the energy loss spectrum with an intensityas high as a few percent from the zero-loss peak. Using this sametechnique, measurements of the electron pulse width can be obtained.

An example of an application of embodiments of the present invention isthe imaging of the non-concerted elimination reaction of dihaloethanes.FIG. 6 is a simplified timing diagram illustrating the use of a methodaccording to the present invention. FIG. 6 illustrates a method of thepresent invention using different electron pulse sequences to isolatethe reactant, intermediates in transition, and product structures. Asillustrated, the specific reaction studied involves the elimination oftwo iodine atoms from the reactant to give the product.

The reactant, for example, diiodoethane, is illustrated at time -t andtime to in FIG. 6A. As illustrated at time -t, the electron pulse 610has been timed to arrive at the sample before the optical initiatingpulse 620. Utilizing the delay stage illustrated in FIG. 1A, the delaybetween the initiation pulse and the electron pulse is varied, enablingimages to be collected at various reaction times. At time to, theoptical initiation pulse 620 and the probe pulse 612 impinge on thesample containing the diiodoethane simultaneously. In the embodimentillustrated in FIG. 6, the initiation pulse is an optical pulse andinitiates the non-concerted elimination reaction of the diiodoethane. Ofcourse, use of the OPO 160 provides for tuning of the optical initiationpulse as desired, for example, for initiation of chemical reactions,spectroscopic analysis, and the like.

FIG. 6B illustrates a snapshot of the elimination reaction at a time t₁after the optical initiation pulse impinges on the sample. At time t₁,the diiodoethane eliminates the first iodine atom as illustrated. Astime progresses, the second iodine atom is eliminated at time t₂ asillustrated in FIG. 6C. The molecular structure of the C₂F₄lintermediate was determined from the frame referencing,diffraction-difference curves ΔsM(t; 5 ps; s). Both the bridged andclassical C₂F₄ 1 structures were considered in the analysis of thediffraction data. The theoretical curves for the classical structuresreproduce the experimental data very well, whereas the fit provided bythe theoretical bridged structure is vastly inferior. Thus, we concludedthat the structure of the C₂F₄ 1 radical intermediate is, in fact,classical in nature, i.e. the iodine atom does not bridge the twocarbons.

Furthermore, we determined that the C—I and C—C distances of the C₂F₄Iintermediate are, respectively, longer and shorter than those of thereactant, while the C—F′ internuclear distance in the radical site(—CF′₂) is shorter than that of the —CF₂I site. These results elucidatethe increased C—C and decreased C—I bond order resulting from theformation of the transient C₂F₄I structure. Moreover, the ≦CCF′ and≦F′CF′ angles become larger than the corresponding angles of thereactant (by ˜9° and ˜12° respectively), suggesting that the radicalcenter (—CF′₂) of the C₂F₄I intermediate relaxes following the loss ofthe first 1 atom. We believe the structures and dynamics reported forthis reaction are important in describing the retention ofstereochemistry in such class of reactions. Furthermore, we believe thisis the first example of resolving such complex structures during thetransition.

In FIGS. 6A-C, the time scale is not drawn to scale, but merely providedto illustrate a series of time-resolved measurements. For thenon-concerted elimination reaction of dihaloethanes time t₁ isapproximately 250 fs and time t₂ is 26±7 ps. Of course, there can beother variations, modifications, and alternatives. Certain methodsaccording to embodiments of the present invention are describedthroughout the present specification and more particularly below.

A method for imaging an object according to an embodiment of the presentinvention may be outlined as follows:

1. Provide a transmission electron microscope comprising a laser source,a cathode, and an electron lens assembly;

2. Form a train of optical pulses, where each of the optical pulses ischaracterized by a Full Width Half Maximum (“FWHM”) pulse length of lessthan 100 fs in duration;

3. Provide a sample (e.g., chemical, biological, physical) for imagingdisposed on a stage assembly;

4. Generate a train of electron pulses by impinging the associated trainof optical pulses on the cathode, where each of the electron pulses ischaracterized by a FWHM pulse length less than 1 ps in duration;

5. Direct the train of electron pulses toward the sample using at leastthe electron lens assembly;

6. Capture a portion of the train of electron pulses using a sensingdevice;

7. Derive information associated with an image of the sample;

8. Process the information associated with the image of the sample;

9. Output a visual representation of the image of the sample using atleast the processed information; and

10. Perform other steps, as desired.

The above sequence of steps provides methods according to an embodimentof the present invention. As shown, the method uses a combination ofsteps including a way of imaging one or more feature of a sample usingone or more pulses of electrons having a short predetermined durationaccording to a specific embodiment. Many other methods and system arealso included. Of course, other alternatives can also be provided wheresteps are added, one or more steps are removed, or one or more steps areprovided in a different sequence without departing from the scope of theclaims herein. Additionally, the various methods can be implementedusing a computer code or codes in software, firmware, hardware, or anycombination of these. Depending upon the embodiment, there can be othervariations, modifications, and alternatives. Further details of thepresent method can be found throughout the present specification andmore particularly below.

FIG. 7 is a simplified flow diagram 700 of an imaging method accordingto an embodiment of the present invention. This diagram is merely anexample, which should not unduly limit the scope of the claims herein.One of ordinary skill in the art would recognize many variations,modifications, and alternatives.

Although the above method has been illustrated in terms of specificsoftware and/or hardware features, it would be recognized that manyvariations, alternatives, and modifications can exist. For example, anyof the hardware features can be further combined, or even separated. Thefeatures can also be implemented, in part, through software or acombination of hardware and software. The hardware and software can befurther integrated or less integrated depending upon the application. Ofcourse, one of ordinary skill in the art would recognize many othermodifications, variations, and alternatives.

A method for imaging an object according to an alternative embodiment ofthe present invention may be outlined as follows:

1. Provide a feature (e.g., 100 nanometers and less) of a sample to beimaged;

2. Place the sample onto a stage assembly;

3. Maintain the sample on the stage assembly in a vacuum environment;

4. Direct one or more pulses of electrons (e.g., one to about 1000electrons per pulse) toward the feature of the sample;

5. Capture a portion of the one or more pulses of electrons, which areassociated with the feature of the sample, using a sensing device;

6. Transfer information associated with the portion of the one or morepulses of electrons associated with the image of the feature of thesample from the sensing device to a processing device;

7. Receive the information associated with the portion of the one ormore pulses of the electrons by the processing device;

8. Process the information associated with the portion of the one ormore pulses of electrons;

9. Output a visual image associated with the feature of the sample usingat least the information associated with the portion of the one or morepulses of electrons associated with the image of the feature of thesample; and

10. Perform other steps, as desired.

The above sequence of steps provides methods according to an embodimentof the present invention. As shown, the method uses a combination ofsteps including a way of capturing an image of a feature of a sampleusing one or more pulses of electrons being directed to the feature anda portion of the electrons captured by a detection device according to aspecific embodiment. Many other methods and system are also included. Ofcourse, other alternatives can also be provided where steps are added,one or more steps are removed, or one or more steps are provided in adifferent sequence without departing from the scope of the claimsherein. Additionally, the various methods can be implemented using acomputer code or codes in software, firmware, hardware, or anycombination of these. Depending upon the embodiment, there can be othervariations, modifications, and alternatives. Further details of thepresent method can be found throughout the present specification andmore particularly below.

FIG. 8 is a simplified flow diagram 800 of an alternative imaging methodaccording to an embodiment of the present invention. This diagram ismerely an example, which should not unduly limit the scope of the claimsherein. One of ordinary skill in the art would recognize manyvariations, modifications, and alternatives.

Although the above method has been illustrated in terms of specificsoftware and/or hardware features, it would be recognized that manyvariations, alternatives, and modifications can exist. For example, anyof the hardware features can be further combined, or even separated. Thefeatures can also be implemented, in part, through software or acombination of hardware and software. The hardware and software can befurther integrated or less integrated depending upon the application. Ofcourse, one of ordinary skill in the art would recognize many othermodifications, variations, and alternatives.

In still a further embodiment, the present invention provides a methodof acquiring time-resolved images using an electron microscope. Suchmethod may be outlined as follows:

1. Provide a feature of a sample to be imaged for a temporalcharacteristic;

2. Place the sample onto a stage assembly of the electron microscope;

3. Maintain the sample on the state assembly in a vacuum environment;

4. Direct one or more first pulses of electrons, each of which has 10 to1000 electrons, toward the feature of the sample;

5. Capture a first portion of the one or more first pulses of electrons,which is associated with a first image of the feature of the sampleduring a first portion of time, using a sensing device during the firstportion of time;

6. Transfer first information associated with the first portion of theone or more first pulses of electrons associated with the first image ofthe feature of the sample during the first portion of time from thesensing device to a processing device;

7. Direct one or more second pulses of electrons, each of which has 10to 1000 electrons, toward the feature of the sample;

8. Capture a second portion of the one or more second pulses ofelectrons, which is associated with a second image of the feature of thesample during a second portion of time, using the sensing device duringthe second portion of time;

9. Transfer second information associated with the second portion of theone or more second pulses of electrons associated with the second imageof the feature of the sample during the second portion of time from thesensing device to the processing device;

10. Process the first information using the processing device;

11. Process the second information using the processing device;

12. Output a first visual image associated with the feature for thefirst portion of time;

13. Output a second visual image associated with the feature for thesecond portion of time; and

14. Perform other steps, as desired.

The above sequence of steps provides methods according to an embodimentof the present invention. As shown, the method uses a combination ofsteps including a way of capturing multiple images of a feature atdifferent time domains according to a specific embodiment. A combinationof one or more ultrafast electron pulses leads to determination oftemporal changes of the feature of the sample according to a specificembodiment. Many other methods and system are also included. Of course,other alternatives can also be provided where steps are added, one ormore steps are removed, or one or more steps are provided in a differentsequence without departing from the scope of the claims herein.Additionally, the various methods can be implemented using a computercode or codes in software, firmware, hardware, or any combination ofthese. Depending upon the embodiment, there can be other variations,modifications, and alternatives. Further details of the present methodcan be found throughout the present specification and more particularlybelow.

FIG. 9A is a simplified flow diagram 900 of yet an alternative imagingmethod according to an embodiment of the present invention. This diagramis merely an example, which should not unduly limit the scope of theclaims herein. One of ordinary skill in the art would recognize manyvariations, modifications, and alternatives.

Although the above has been illustrated in terms of specific softwareand/or hardware features, it would be recognized that many variations,alternatives, and modifications can exist. For example, any of thehardware features can be further combined, or even separated. Thefeatures can also be implemented, in part, through software or acombination of hardware and software. The hardware and software can befurther integrated or less integrated depending upon the application. Ofcourse, one of ordinary skill in the art would recognize many othermodifications, variations, and alternatives.

In yet an alternative specific embodiment, a method for capturinginformation from one or more samples using electron beam pulses isbriefly described below.

1. Provide a feature of a sample to be imaged;

2. Place the sample onto a stage assembly;

3. Maintain the sample on the stage assembly in a vacuum environment;

4. Irradiate a cathode using one or more pulses of electromagneticradiation;

5. Direct one or more pulses of electrons, each having about 10 to about1000 electrons, toward the feature of the sample derived from theelectromagnetic radiation;

6. Capture a portion of the one or more pulses of electrons, which isassociated with a characterization of the feature of the sample, using asensing device;

7. Transfer information associated with the portion of the one or morepulses of electrons associated with the characterization of the featureof the sample from the sensing device to a processing device;

8. Process the information;

9. Output one or more indications associated with the feature of thesample using at least the information associated with the portion of theone or more pulses of electrons associated with the image of the featureof the sample; and

10. Perform other steps, as desired.

The above sequence of steps provides methods according to an embodimentof the present invention. As shown, the method uses a combination ofsteps including a way of identifying a character of a feature of asample using one or more pulses of electrons, which is used with adetection device, e.g., CCD array. Depending upon the embodiment, thepresent method can be used for imaging, diffraction, and other analysistechniques. Many other methods and system are also included. Of course,other alternatives can also be provided where steps are added, one ormore steps are removed, or one or more steps are provided in a differentsequence without departing from the scope of the claims herein.Additionally, the various methods can be implemented using a computercode or codes in software, firmware, hardware, or any combination ofthese. Depending upon the embodiment, there can be other variations,modifications, and alternatives. Further details of the present methodcan be found throughout the present specification and more particularlybelow.

In some embodiments of the present invention, methods for determiningtemporal characteristics of one or more feature of objects using anelectron microscope assembly are provided. Merely by way of example, asample may be imaged at various times during a transition betweenmultiple states. FIG. 9B is a simplified timing diagram illustratingpulse trains according to an embodiment of the present invention. Duringtime period 920, a number of electron pulses 922 are provided as a pulsetrain. In a particular embodiment, the temporal width of each pulse is100 fs and the delay between adjacent pulses is 12.5 ns. Additionally,each of the pulses are referenced to a baseline time, for example thearrival at the sample of separately provided initiating pulses. In oneembodiment, sensing elements, for example, a CCD camera, are activatedduring time period 920 to acquire an image resulting from interaction ofelectrons in the pulse train with the sample. As an example, theunfolding of a protein is imaged using systems according to anembodiment of the present invention. The unfolding process is activatedby a pump optical pulse. As described above, the timing between the pumpand electron pulses is selected to provide for the collection of imagesat a given time during the unfolding process. As an example, the pulses922 are delayed from the pump pulse by 100 ps in one embodiment. Duringthe time between adjacent pulses, for example, 12.5 ns, the proteinfolds back to the steady state condition. Upon initiation by asubsequent pump pulse during time period 920, the unfolding process isrepeated and an additional electron pulse interacts with the sampleafter the same delay time. In embodiments in which time period 920 is onthe order of a second, millions of electron pulses with the same delaytime interact with the sample, resulting in the collection of a firstimage. In some embodiments, the time period 920 is on the order ofseveral seconds. One of ordinary skill in the art would recognize manyvariations, modifications, and alternatives.

A second time period 930 is illustrated in FIG. 9B. In anotherembodiment, the delay time between the initiating pulse and the pulses932 is a second delay time, for example, 200 ps. In the embodimentillustrated in FIG. 9B, the process described above is repeated,acquiring an image of the unfolding process at a second time during theunfolding process, for example 200 ps. As illustrated, additional timeperiods 940 are provided in some embodiments, producing, in thecombination, sequential, time-resolved images of the sample. In someembodiments, there are n additional time periods, with n being apredetermined number.

FIG. 9C is a simplified flowchart illustrating image collectionaccording to an embodiment of the present invention. In step 950, animage of a portion of a sample is acquired with the sample in state 2.In step 955, an additional image of a portion of a sample is acquiredwith the sample in state 2. In step 960, n additional images areacquired with the sample in n additional states. Using methods andsystems according to the present invention, ultrafast electron pulsesused in acquiring images are characterized by low-electron content.These electron pulses reduce pulse broadening in both time and space andprovide low electron fluences, as is beneficial in minimizing damageduring the imaging of biological samples. In embodiments of the presentinvention, the number of pulses included in each of the time periods isa predetermined number. Generally, the selection of the number of pulsesbalances the number of electrons desirable for imaging, the damage tothe sample, the time required to collect the image, and the like.

FIG. 10 is a simplified flow diagram 1000 of a still an alternativeimaging method according to an embodiment of the present invention. Thisdiagram is merely an example, which should not unduly limit the scope ofthe claims herein. One of ordinary skill in the art would recognize manyvariations, modifications, and alternatives.

Although the above method has been illustrated in terms of specificsoftware and/or hardware features, it would be recognized that manyvariations, alternatives, and modifications can exist. For example, anyof the hardware features can be further combined, or even separated. Thefeatures can also be implemented, in part, through software or acombination of hardware and software. The hardware and software can befurther integrated or less integrated depending upon the application. Ofcourse, one of ordinary skill in the art would recognize many othermodifications, variations, and alternatives.

Although certain figures have been described in terms of a specificembodiment, one of ordinary skill in the art would recognize manyvariations, alternatives, and modifications. Of course, there can avariety of variations without departing from the scope of the claimsherein.

To prove the principle and operation of the present invention, weperformed experiments for certain applications of embodiments of thepresent invention. The present invention used the present ultra fastelectron microscope system, which has been previously described. Thatis, the ultrafast system includes various parameters such as one or moreultrafast pulses of light to generate one or more ultrafast pulses ofelectrons according to a specific embodiment. Although these parametershave been used, there can be many other variations, modifications, andalternatives.

In a specific example, we have studied interfacial molecular assembliesat the nanometer scale, which is of importance to chemical andbiological phenomena using the present methods and systems. For water,the directional molecular features of hydrogen bonding and the differentstructures possible, from amorphous to crystalline, make the interfacialcollective assembly on the mesoscopic scale much less understood beforeour discoveries. Structurally, the nature of water on a substrate isdetermined by forces of orientation at the interface and by the netcharge density, which establishes the hydrophilic or hydrophobiccharacter of the substrate. However, the transformation from ordered todisordered structure and their coexistence critically depends on thetime scales for the movements of atoms locally and at long range.Therefore, it is desirable to elucidate the nature of these structuresand the time scales for their equilibration.

In an experiment performed using an embodiment of the present invention,we made a direct determination of the structures of interfacial waterwith atomic-scale resolution, using diffraction and the dynamicsfollowing ultrafast infrared (IR) laser-initiated temperature jump.Interfacial water is formed on a hydrophilic surface (silicon,chlorine-terminated) under controlled ultrahigh vacuum (UHV) conditionsas illustrated in FIG. 11. With the atomic-scale spatial, temporal, andenergy resolutions provided by embodiments of the present invention, theevolution of non-equilibrium structures was monitored, their ordered ordisordered nature was established, and the time scale for the breakageof long-range bonding and formation of new structures was determined. Asdiscussed below, we identified the structured and ordered interfacialwater from the Bragg diffraction and the layered crystallite structurefrom the Debye-Scherrer rings. The temporal evolution of interfacialwater and layered ice after the temperature jump was studied withsubmonolayer sensitivity. We compared these results with those obtainedon hydrophobic surfaces, such as hydrogen-terminated silicon or silversubstrate.

FIG. 11 illustrates the structure of water at the hydrophilic interface.As illustrated, the chlorine termination on a Si(111) substrate forms ahydrophilic layer that orients the water bilayer. The closest packingdistance (4.43 Å) between oxygen atoms in the bottom layer of water issimilar to the distance (4.50 Å) between the on-top and interstitialsites of the chlorine layer, resulting in specific bilayer orientations(±30°) with respect to the silicon substrate. This ordered stackingpersists for three to four bilayers (˜1 nm) before disorientation takesplace and results in crystallite islands, forming the layered structure.As illustrated, the size of atoms is not to scale for the van der Waalsradii.

Spectroscopic techniques, such as internal reflection and nonlinear(e.g., second-harmonic generation and sum-frequency generation (SFG))optical methods, are sensitive to surface molecular changes. Forexample, the presence of polar ordering of ice films on a Pt(111)surface was shown to have a decay length of 30 monolayers, and transientSFG response from D₂O ice crystals on a CO/Pt(111) surface has indicatedthe presence of melting and recrystallization without desorption. Here,structures were determined using diffraction with ultrafast timeresolution, providing a spatial resolution of 0.01 Å. We can monitor thechange on selective internuclear distances of the hydrogen bondingnetwork, e.g., those of OH—O and O—O at 2.75 Å and 4.5 Å, respectively.Unlike previous studies on ultrafast surface restructuring andsubnanosecond melting, we can probe supramolecular structural dynamicson surfaces and observe clear separation of the diffraction of theinterfacial water from that of the substrate.

Using embodiments of the present invention, water was prepared on asingle crystal Si(111) surface terminated chemically with chlorine atomsto make a hydrophilic interface. The crystal was mounted on a goniometerwith an angular precision of 0.005° in a ultrahigh vacuum (UHV)environment. The crystal can be cooled to a temperature of 100K or othersuitable temperature. The layer preparation on the surface generallyrequires characterization of the substrate by low-energy electrondiffraction and Auger spectroscopy, as well as in situ monitoring oflayer growth by reflective high energy electron diffraction. In ourcase, the electron flux (˜1 pA/mm²) was relatively small, so damage andcharging of the molecular layers was reduced. The photon pulses producedby the femtosecond laser (typically ˜1 mJ, 800 nm, and 120 fs at a 1 kHzrepetition rate) were directed at a 30° angle into the scatteringchamber and focused on the substrate to initiate the temperature jump.

A weaker beam was split from the photon pulse beam, frequency tripled ina specific embodiment, (˜10 nJ at 266 nm), and focused onto aback-illuminated silver photocathode after an adjustable time delay togenerate the electron pulses via the photoelectric effect. The electronpulses in this embodiment have a de Broglie wavelength λ=0.07 Å at 30keV. A series of deflectors and apertures were used to guide theelectron beam for an incidence to the surface of θ_(i)<1°, which istypically appropriate for high sensitivity on nanometer-scale surfaceassemblies. The arrival of the electron pulses was controlled to definea sequence of images that were recorded with a low-noise,image-intensified, charge-coupled device (CCD) camera assembly capableof single-electron detection.

We first characterized the diffraction of the Si substrate before dosingwith water. By rotating the crystal, we obtained the rocking curves andthe dependence of diffraction pattern on θ_(i). These diffractionpatterns represent the intersection between Ewald's sphere and thereciprocal lattice defined by the substrate. Thus, the momentum transfercoordinates (s), defined by s=4π/λ·sinθ/2, where the scattering angle,θ, can be mapped out for any given diffraction image at the incidenceangle θ_(i), in situ zero-of-time was also determined using thesubstrate surface temperature jump. As interfacial ice was forming onthe surface at 110 K, the diffraction images showed the transition fromthe Bragg spots of the substrate to the new spots and ringscharacteristic of water. After the substrate temperature jump, we useddifferent sequencing of electron pulses in order to separately imageevolving structures of water. This diffraction difference method allowsfor the isolation of the only transient structure involved because thereference time (t_(ref)) can be chosen before or after the arrival ofthe initiating pulse, or different times during the change.

FIG. 12 shows the diffraction images obtained with ultrashort electronpulses, but without the initiating laser pulse (equivalent to t_(ref) atnegative time). The processes of in situ growth of ordered ice isillustrated through vapor deposition of water on a cold (110 K) siliconsubstrate is illustrated. The adsorption of water on the substrate isseen from the disappearance of 111 Bragg spot of silicon (see, forexample, FIG. 12A) and the formation of 111 Bragg spot of crystallineice, together with the diffraction rings of amorphous ice (see, forexample, FIG. 12B). Annealing promotes the formation of long-rangecrystalline structure, as shown in the increase of the brightness of thespots and the sharpening of the rings (see, for example, FIGS. 12C andD). The structures stabilize as the diffraction shows nearly no changein the rings, spots, and streak (see, for example, FIGS. 12E and F).FIGS. 12G and 12I show experimentally observed diffraction andsimulations of spots from a nanometer-thick substrate, orientated cubic(Ic) and hexagonal (Ih) structures. FIGS. 12J shows experimentaldiffraction rings when radially averaged in s space produceone-dimensional diffraction intensity curves. FIGS. 12K and L showtheoretical diffraction intensity curves, with the peaks identified withBragg reflections. The clear distinction of different orderings and theearly appearance of spots (not rings) in the annealing suggestheterogeneous layers on the surface with the crystallites layered by theordered interfacial water. Moreover, the diffraction spots are sharp,which indicates that the interfacial water posses well-definedorientation; in contrast, the diffraction rings are circular, which iscompletely in agreement with randomly orientated crystallites.

As illustrated, the diffraction pattern is composed of rings, spots, andstreaks. The disappearance of substrate diffraction and the appearanceof surface water diffraction as monolayers of ice formed after annealingare evident. The observed Bragg spots indicate that water molecules areoriented by the substrate with a long-range order. The rings coincidewith the spots in the reciprocal space (s space), and these ringsindicate the emergence of crystallite structure: islands of orderedwater, but disordered in orientation. The rings are sharp enough todefine a crystallite that is not amorphous. The structural evolution ofthe interfacial water layers as a function of temperature was calibratedat near-equilibrium condition (temperature ramp, ˜2°/min). We found thatat this nanometer scale the crystallization of the initially depositedamorphous ice begins near 140 K and reaches the saturation at ˜150 Kwith the highest degree of long-range order. The sublimation of thewater layers occurs at 157±1 K. From both Bragg spots and the rings, wecan determine the structure by comparing these diffraction images withthose predicted by the symmetry of ice lattices.

The intensity of diffraction rings when plotted against s gives thecorresponding peaks of Bragg reflections. The theoretical diffractionpatterns for both cubic and hexagonal structures are shown in FIGS. 2Kand L. These plots were obtained by summing the phases of athree-dimensional (3D) crystallite (a cell of a dimension of 5 nm ineach direction) and averaging over all orientations. The peaksillustrated in FIG. 12J agree well with reflections from the 111, 220,and 311, etc., planes for cubic ice (see, for example, FIG. 12K), whichis a dominant structure [for comparison, see hexagonal ice diffraction(see, for example, FIG. 12L)]. From the s values in FIG. 12J, wedetermined the interplanar distances to be 3.80±0.23 Å, 2.27±0.15 Å, and1.93±0.07 Å for the (111), (220), and (311) planes, respectively,consistent with reported powder diffractions of cubic structure. Theuncertainties are governed by the observed width of the diffractionpeaks.

The surface-oriented water is a well-defined crystalline structure(i.e., epitaxially grown from the substrate), and this structure isresponsible for the sharp Bragg spots. We reproduced theoretically theposition and relative intensities of Bragg spots as illustrated in FIG.12H by summing phases of long-range ordered interfacial water (10 nm by10 nm wide; 1.5-nm-thick layer). For this hydrophilic substrate, wefound that the closest packing distance between oxygen atoms in thebottom layer of water (4.5 Å) is similar to that between on-top andinterstitial sites (4.43 Å) of the chlorine layer. This makes possiblethe long-range packing of the water bilayer on the surface and leads tothe unique 30° rotations with respect to the substrate layer. Thus thetwo-dimensional (2D) surface unit cell of water can be described as asuperlattice as illustrated. This assignment was derived directly fromthe symmetries and positions of Bragg spots of the substrate and thoseof water. Inspection of the Bragg spots of ice identifies the twodomains (±30° rotations) in the satellites of the main Bragg (i.e., 111)peak; one set is formed by 022, 111, 311; the other is by 202, 111, 131(see FIG. 12H). Water interacts with the substrate at two sites, likelythrough its oxygen in the interstitial site with sp³ hybridized orbitalsoverlapping with three chlorine atoms, or through hydrogen sitting ontop of the chlorine atom.

From the diffraction results, we established that water is structured onthe hydrophilic surface mainly as cubic (see the comparison betweentheory for orientated cubic (Ic) and orientated hexagonal (Ih), and theexperiment as illustrated] and is different from structures (hexagonal)found on Pt(111) substrate. Our theoretical modeling of the position ofthe 111 Bragg spot gives an interlayer spacing of 3.66±0.26 Å, entirelyconsistent with the value obtained from the rings (3.80±0.23 Å). Theapparent brightness and width of Bragg spots are an inherent reflectionof the size of interferences, and from them we obtained a thickness ofnanometer scale, which is also consistent with theory.

FIG. 13 shows data collected in an experiment performed using anembodiment of the present invention. In FIGS. 13A and B, diffractiondifference images at negative (−30 ps) and positive (100 ps) times areillustrated. The t_(ref) is at −70 ps in FIGS. 13C to H. The radiallyaveraged diffraction difference intensity curves at several delay times((C) −30 ps, (D) 10 ps, (E) 20 ps, (F) 100 ps, (G) 530 ps, (H) 1130 ps)show the structural dynamics for layered crystallites at substrateenergy fluence of 22 mJ/cm². Note the depletion (negative difference)and the increase (positive difference) at well defined s values. FIG.13I shows the diffraction difference images for the 111 Bragg spot. Thevertical axis is s in the reciprocal space, and the horizontal axis isthe azimuthal scattering angle.

For the dynamics of the system, we followed the diffraction as afunction of time after the temperature jump of the substrate asillustrated in FIG. 13. When t_(ref) is at negative time (−70 ps), theimage at −30 ps (referenced to the −70 ps, see FIG. 13A) shows nodiffraction difference intensity, as expected. At positive time asillustrated in FIG. 13B and 13I, both the Bragg spots and rings emerge,but with striking structural changes (note the displacement of rings andspots). The different panels display the evolution with clear indicationof the disappearance of the “old” structure and the appearance of a“new” structure. However, the behavior is similar in appearance to thatof a “phase transition”: at short times (10 ps (FIG. 13D) and 20 ps(FIG. 13E)], we observed depletion of old structure, whereas atintermediate times (FIGS. 13F and 13G), there appeared a region ofcoexistence of disordered and crystal-like water. At very long time(FIG. 13H), the system reverted to the original structure, but with somedifference in bond distances. It should be noted that in a separatecryocooling experiment, we changed the substrate temperature by rampingat near-thermodynamic equilibrium condition, just below sublimationtemperature (157 K), and found the ice structure to be in agreement withthose obtained after restructuring at long times. This behavior for thecrystallites away from the surface (rings) contrasts that of thestructured, crystal-like water on the surface (spots). FIG. 13I showsthe evolution of one spot with time that exhibits the sametrend—depletion and restructuring—but the dynamics are very different.

FIG. 14 illustrates the temporal evolution of diffraction gated in the111; reflection region. As shown in FIG. 14A, the early-time (≦100 ps)depletion of old 111 diffraction spot and ring at several energyfluences. In FIG. 14B, the formation of new 111 diffraction spot andring, also at early times. Note the apparent delays with respect to thezero-of-time, which is independently determined with uncertainty of 3ps. The corresponding changes at longer times are shown in FIGS. 14C and14D.

By examining the rate of change at different temperatures, which iscontrolled by changing the fluence of the heating pulse of thesubstrate, we found, as illustrated in FIG. 14, that gating in the 111reflection region shows a depletion of the old peak (see FIGS. 14A and14C) and a corresponding buildup (see FIGS. 14B and 14D) of a new peak.This behavior mirrors the breakage of old bonds (by melting) and theformation of a new structure. The melting of the layered crystallitesfollows the laser excitation at the substrate surface within 5 ps. Incontrast, from the same data, images gated on the 111 Bragg spot had adelay time of 36±3 ps for interfacial water at the same energy fluence(22 mJ/cm²). Because water does not absorb light directly at 800 nm, therelatively prompt response of layered crystallites signifies the highefficiency of heating by nondiffusive vibrational couplings on thisultrashort time scale; if diffusional, the layered ice should melt afterthe interfacial one. As the fluence increases, the delay decreased forinterfacial water, but the time constants for depletion remained similarat 37±5 ps. The results suggest the presence of a higher energy(friction) barrier for the structured water caused by the long-rangeorder and dipolar orientation force of the hydrophilic substrate.

These observations indicate that at the highest temperature of thesubstrate (fluence, 42 mJ/cm²), the interfacial water continues to loselong-range order and that only when the maximum change is reached willrestructuring begin as illustrated in FIGS. 14A and 14B. However, nearthe maximum change, the new structure begins to form in the region ofcoexistence, followed by restructuring (see FIGS. 14C and 14D). In therestructuring at long times, the dissipation of energy (cooling) occursthrough redistribution and heat diffusion. In this regime, from thesolution of the heat diffusion equation, we estimate a surfacetemperature of ˜150 K at 1 ns; the temperature at maximum change is ˜370K. Over the entire time scale of structural change, thermal desorptionwas found to be insignificant because we observed the recovery ofdiffraction rings and spots back to nearly their original intensities,as shown in FIG. 14A from the constancy of the baselines at negativetimes for all fluences. The lack of effective desorption, as also foundfor ice on CO/Pt surface, reflects the difference between desorption atnear-equilibrium or far-from-equilibrium temperatures. The appearance ofthe plateau region at the higher fluence (42 mJ/cm²) for the depletionof old structure and the making of new structure, as well as thecoexistence of old and new structures, suggests the involvement ofcollective modes in restructuring, analogous to phase transitions.

FIG. 15 illustrates the radial distribution function (RDF) for Ic isshown with the internuclear distance density. The local distances at2.75 Å (OH—O) and 4.5 Å (O—O) in a diamond tetrahedron unit are markedfor the comparison with data (FIG. 15B to E). The experimentaldifference RDF curves were obtained by sine transform from thecorresponding difference intensity curves (see FIG. 13). Changes ofdistance densities are evident at the three positive times, whereas nochange is observed at negative time. The light gray curve in the panelsis the Ic curve, scaled and superimposed for comparison. The change inthe RDF clearly shows the depletion of the old structure and formationof the new structure (as in FIG. 13) but here identifies the bondsinvolved. In FIG. 15F, the corresponding structural changes at −5, +10,and +1130 ps as illustrated.

In supramolecular systems, such as the one discussed here, the localstructures within a unit cell, in addition to the long-range order, canbe examined by inverting (Fourier transform) the diffraction curves tothe real space. FIG. 15A to E illustrates such obtained radialdistribution functions [f(r)] reveal the densities of internucleardistances. For cubic ice, the second nearest O—O distance (4.50 Å)correlates with the hydrogen bond OH—O distance (2.75 Å) in a diamondtetrahedron. The temporal changes of the density of these two distancesthus provide the dynamics at the local molecular level of therestructuring of the hydrogen bond network. At negative time (−5 ps, seeFIG. 15B), no change of density is observed, as expected. At 10 ps (FIG.15C), depletion of O—O peak is observed, although the change for theOH—O peak is insignificant. This depletion indicates the rupture of thenetwork (to amorphous) in 10 ps. At 150 ps delay (FIG. 15D), significantbut shifted depletions coexist with emerging new distances. At thelongest delay, 1130 ps (FIG. 15E), the original cubic-like structure isrecovered, as evidenced in the reduction in the diffraction difference,but the new structure is still slightly “hot” due to slow rate ofdiffusion (ns to μs). For such structures, we determined the interplanardistances of 4.22±0.37 Å (111 plane), 2.42±0.20 Å (220 plane), and1.97±0.05 Å (311 plane). A structural picture of local molecular changesis depicted in FIG. 15F.

Interfacial water on the hydrophilic surface substrate has distinctivestructures and dynamics compared with those of water layers onhydrophobic surfaces. The time scale for the breakage of long-rangeorder of the interfacial layer (37 ps) is an order of magnitude longerthan that for breaking hydrogen bonds in bulk liquid water, and thelocal OH—O and O—O bond distances from diffraction are directly involvedin the change. These results suggest that the time scale for energy flowin the assembled water structure is much shorter than that of energylocalization for desorption of individual molecules. It should be notedthat the maximum transient temperature is 370 K; the equilibriumdesorption temperature is 157 K. Moreover, the restructuring timeinvolving long-range order is longer than the time for amorphization, aprocess in which the O—O correlation is lost before the OH—Ocorrelation. Perhaps it is not accidental that the time scale for losingthe hydrogen bond network (37 ps) is similar to that reported forinterfacial water near hydrophilic protein surfaces (20 to 50 ps) and isvery different from that of bulk water (700 fs-1.5 ps). In separateexperiments, we also studied hydrophobic surfaces, such ashydrogen-terminated and silver-coated silicon substrates, and found thatinterfacial ordering has changed into a distribution around the (110)orientation for the former and is absent for the latter substrate.

Therefore utilizing embodiments of the present invention, it ispossible, with unprecedented resolutions and sensitivity, to studynanometer-scale supramolecular structures, along with those of amorphousand crystalline solids. This demonstrates the diverse applicationsavailable using embodiments of the present invention, in particular forthe probing of interfaces and surfaces with atomic-scale resolution. Ofcourse, these results are merely examples, which should not unduly limitthe scope of the claims herein. One of ordinary skill in the art wouldrecognize many variations, modifications, and alternatives.

In another experiment, we demonstrated the potential of methods andsystems according to the present invention for the direct determinationof surface structural dynamics of crystalline solids (GaAs), followingimpulsive femtosecond laser excitation. From the change of Braggdiffraction (shift, width, and intensity), we show in the discussionthat follows, by direct inversion of the diffraction data, thatcompression and expansion of the atoms occur on the −0.01 Å to +0.02 Åscale, respectively, and that the transient temperature reaches itsmaximum value (1565 K) in 7 ps. The onset of structural change lagsbehind the rise in the temperature, demonstrating the evolution ofnon-equilibrium structures. These structural dynamics results arecompared with those of nonthermal femtosecond optical probing, and theagreement for the temperature response from the fluence dependence ofthe dielectric function is impressive. The success in the directobservation of surface (monolayers) structural dynamics with combinedultrafast temporal and atomic-scale spatial resolutions promises manynew applications for embodiments of the present invention.

GaAs is an ideal system to demonstrate this potential of embodiments ofthe present invention for surface studies, especially as its crystallineand semiconducting properties are well-quantified. This has allowed awide range of ultrafast optical experiments that vary from the probingof carrier properties to electronic disordering or change in symmetry.In addition to these optical studies, recent ultrafast X-ray experimentson GaAs revealed bulk lattice dynamics following femtosecond laserexcitation. However, these ultrafast X-ray experiments could not probethe surface owing to the large penetration depth into the crystal byX-rays, typically up to several μm. On the other hand, opticaltechniques that probed the surface on the scale of a few nanometerscould not directly determine the structure with atomic-scale resolution,but gave valuable information on the response of the dielectric functionand lattice disordering. The large scattering cross-section of electronscombined with ultrafast time resolution allows the bridging of this gapin addressing the dynamics of surface structures in real time.

The experiments described below were performed using the systemsillustrated, for example, in FIG. 1. Under ultrahigh vacuum (typically10⁻¹⁰ Torr), and following surface characterization by low-energyelectron diffraction and Auger spectroscopy, the sample was brought tothe scattering position where beams of the laser-pulse excitation andelectron-pulse probe intersected in space, with an adjustable time delayAt (the zero of time was determined in situ utilized methods describedabove). In some embodiments of the present invention, the chamberincludes sputtering and cleaning tools. We terminated semiinsulatingGaAs (111) crystals by a monolayer of chlorine with a Cl atom atop eachGa atom, saturating the otherwise dangling bond of Ga. The surfaceretained its integrity throughout the experiments, as evidenced by theunchanged quality of the diffraction patterns (symmetry, spot profiles,and intensities), as described more fully below. The crystal, placed ona goniometer with three degrees of freedom in translation and two axesof rotation, was positioned in space with a precision of 0.01° inrotation, and of 10 μm in translation. The experiments were carried outat room temperature.

FIG. 16 is a conceptual diagram illustrating an embodiment of thepresent invention and the structure of the chlorine-terminated GaAs(111) crystal. The output of a Ti:Sapphire femtosecond amplifier (120fs, 800 nm, 2

mJ, 1 kHz) was frequency tripled to yield a beam at 266 nm (400 μJ, <300fs, 1 kHz). This UV beam was brought to the scattering chamber toprovide the initial heating pulse at t=0. A very small fraction of thisbeam was directed synchronously onto a back-illuminated Ag photocathodeto generate the electron pulses. These electron pulses, afteracceleration and focusing, were guided to the crystal, where theyoverlapped in space and time (delay At) with the heating pulse. Althoughelectron pulses were made as short as 500 fs, in this grazing incidence,the spatial extent decreases this resolution to tens of ps. However,because of the sensitivity achieved (20 s per frame), we were able toreduce the experimental temporal cross-correlation to 7 ps; withconvolution and at the level of signal reported herein, we readilyobtained a 1-2 ps response. This was made possible by reducing thespatial extent of the substrate to 400 μm by masking techniques,resulting in a transit time of 4 ps. The sample, placed on ahigh-precision five-axis goniometer, was positioned to allow theelectron beam to impinge at the chosen incidence angle (θ₁;<5°),azimuthally along the <112> direction. In the Figure, L0 and L1 refer tothe zero-order and first-order Laue zones. The resulting diffractionpatterns were recorded in the far field by an imaging CCD camera system.In the inset to FIG. 16, the structure of the crystal is shown, with thebilayer spacing of 3.26 Å.

Femtosecond laser heating at t=0 initiated the dynamics, which weresubsequently probed by an ultrashort packet of electrons of 30 keV(λ_(deBroglie)≈0.07 Å). The electrons impinged the surface at a smallincidence angle (θ_(i)≈1°) and at this grazing incidence, reflectionhigh-energy electron-diffraction methods are uniquely suited and havebeen used for studies of superheating with a temporal resolution of ≈100ps. The electron pulses were generated by a modified Williamson-Mouroustreak camera arrangement. In the system utilized for this experiment,we obtained pulses of duration as short as ≈500 fs, but for the grazinggeometry of the experiment, the temporal cross-correlation was 7 ps. Bycontrolling the arrival of the electron pulse (Δt in FIG. 16), we canprobe the structure prior to or following the heating pulse excitation.Structural changes were followed in real time by monitoring the Braggreflections and rocking curves, as recorded by a CCD (charge-coupleddevice) imaging assembly capable of single electron detection. Threefeatures of the diffraction were followed as a function of time: theBragg peak shift, width, and intensity.

FIG. 17 (A and B) illustrates static diffraction images of the crystal(111) surface obtained by the ultrashort electron pulses without timeresolution. In FIG. 17A (reference letter a), a diffraction imageshowing the intense (0,0) in-phase reflection, together with the streaksand spots in the Laue zones is illustrated. FIG. 17B (reference letterb) illustrates an experimental rocking curve for the (0,0)reflection-(111) lattice planes. As one of skill in the art willappreciate, the periodicity allows the unequivocal identification of theBragg reflections and gives the interlayer spacing.

FIG. 17 (A and B) presents a typical static diffraction image, whichdisplays the very strong (0,0) and other Bragg reflections. FIG. 17Ashows a diffraction pattern, for which the incidence angle was tuned toreveal higher-order diffraction peaks, as well as diffraction streaks inthe zero-order Laue zone. These and similar data allow the precisedetermination of the camera distance from the scattering position (170±1mm), by direct inversion of the angular separation between the streaksat low angles, or from the Bragg spots in the higher Laue zones. Thisinversion is direct because the lattice rods are separated in reciprocalspace by an in-plane inverse distance of 3.14 Å⁻¹ along the intersectionwith the Ewald sphere of radius 2πλ⁻¹=90 Å⁻¹. By gating the detection onthe (0,0) Bragg spot and following the diffraction position as afunction of the incidence angle, we also obtained the experimentalrocking curve, which gives the GaAs lattice periodicity along the (111)direction (n=1, 2, . . . ). This is shown in FIG. 17B, where theincidence angle was varied over several degrees. The experimentalperiodicity in θ_(i) of 0.60°±0.02° is in quantitative agreement withthe expected value of 0.61° obtained for the lattice bilayer spacing of3.26 Å.

FIG. 18 shows the time dependence of the Bragg reflection centerposition, intensity, and width, following the laser heating excitationat t=0. In FIG. 1 8A, the center position of the Bragg spot as afunction of time and fluence is plotted. The vertical axis on the rightgives the angular deviation and the left axis shows the correspondingchange in lattice spacing, perpendicular to the (111) surface plane. Acontraction takes place at early times (Δd<0), followed by latticeexpansion (Δd>0). The inset in FIG. 18 shows the evolution at a longtime. In FIG. 18B, a comparison of the integrated intensity of the Braggspot (temperature) with the change in lattice spacing for the data setobtained at 2 mJ cm⁻² is illustrated. The right axis gives the ratio ofthe time-dependent integrated intensity of the Bragg spot (I) to itscounterpart at negative delay (I_(o)) on a logarithmic scale, from whichthe temperature scale is obtained. The lattice expansion is also shownwith its scale on the left axis, together with the broadening of theBragg spot, depicted by the dashed line. The apparent delay between thetemperature rise and the lattice expansion is noted by the two arrows asdescribed below.

In the time-resolved experiment, depicted schematically in FIG. 16, theexcitation pulse at t=0 defines the initial temperature and structuralchange. In FIG. 18, we follow the center position and intensity of then=2 (0,0) Bragg spot as a function of time. We also monitor the width,as shown in FIG. 18B. FIG. 18A presents the change in the peak centerposition, which maps out the change in lattice spacing in the (111)direction. Results are shown for fluences of 9% and 45% of theexperimentally determined 4.5 mJ cm⁻² damage threshold at 266 nm. Theangular deviation (Δθ) of the Bragg spot directly gives the change inlattice spacing (Δd₁₁₁), from the relation Δd₁₁₁=−Δθ·d₁₁₁·{2 sin(θ/2)}⁻¹(θis the total scattering angle). A deviation to larger or smallerangles (Δθ>0 or Δθ<0) is therefore the signature of lattice contractionor expansion.

From the results shown in FIG. 18A, it is evident that the top surfacelayers of the crystal immediately contract following excitation at t=0.The amplitude of this initial contraction is given for two fluences, butthe complete fluence dependence is presented in FIG. 19. After theinitial contraction (−0.015 Å), the system expands to a maximumamplitude (+0.025 Å), which strongly depends on the fluence: the largerthe fluence the more ample the expansion. The data also show that boththe onset time and the velocity of the expansion (≈m s⁻¹) stronglydepend on the fluence: expansion occurs earlier and faster at higherfluences. After reaching its maximum expansion, the system contractsagain toward the original lattice spacing on a much longer time scale,beyond 50 ps, but a finite smaller expansion persists for at leastseveral nanoseconds. Observations were also made for 800-nm femtosecondexcitation at various laser fluences and the behavior is similar, thatis, an initial contraction, followed by an expansion and subsequentreturn toward the initial lattice spacing. This similarity in form,compared to that in the 266-nm experiment, indicates that the observedstructural dynamics is not dominated by a charging of the surface byphotoemission, as excitation at 800 nm and/or at low fluences results ina similar behavior.

FIG. 19 illustrates the fluence dependence of the structural dynamicsdemonstrated by the experiment. In FIG. 19A, experimental traces for aset of data at the indicated fluences are illustrated. In FIG. 19B, theamplitude at maximum change is shown as a function of excitationfluence. The time-dependence is displayed in the inset (1:0.04, 2: 0.2,3:1, 4:3 mJ cm⁻²). Notably, the temporal resolution is lower than thatin FIG. 18, because these fluence-dependent measurements were madewithout masking in order to explore the lowest possible range offluences.

The transient temperature is evident in the change in thediffraction-integrated intensity with time. This is presented in FIG.18B, for excitation at 45% of damage threshold (2 mJ cm⁻²), by plottingthe evolution of the integrated intensity of the Bragg spot as afunction of time. Using the tabulated Debye-Waller factors for bulkGaAs, and taking into account the two-dimensionality of the surface, weobtained an initial temperature jump to 1565±83 K. The system cools downon the timescale of a few hundred picoseconds to reach ≈510 K after 1ns. The initial temperature jump has a rise time of 7 ps (10 ps beforedeconvolution), in perfect agreement with results from femtosecondoptical studies of the dielectric function. Moreover, the maximumtemperature reported above is close to the value (1300-1500 K)extrapolated from these optical studies at corresponding fluence. Forthe lower fluence regime (0.4 mJ cm⁻²) presented in FIG. 18A, we find atemperature jump to 420±18 K, with a decay leveling off at 320±5 K after1 ns. In this case too, the rise time and the maximum temperature areconsistent with the optical study.

The evolution of the lattice expansion and that of the temperature arejuxtaposed in FIG. 18B, together with the width. Strikingly, thetemperature evolution precedes the lattice expansion, and we measured adelay of 15 ps between the rise in temperature and the structuralexpansion. This lag in structural expansion provides direct evidence forthe proposed delayed lattice changes following an impulsive initialtemperature. We note that the temperature jump to 1565±83 K is similarto (or even exceeds) the 1513 K melting point, whereas the excitationfluence is only half of the damage threshold. However, as evident fromFIG. 18B, the peak temperature does not persist for a long time and thesystem does not lose its crystalline structure.

The lagged structural change reaches its maximum expansion of +0.025 Åat a temperature of ≈1000 K (see FIG. 18B). This lattice expansion inthe nanometer-scale structure may now be compared with the expansion inbulk GaAs. From the linear expansion coefficient of bulk crystals, atemperature of 1000 K would correspond to a linear lattice expansion of0.013 Å, and this value differs by a factor of 2 from our experimentalvalue of 0.025±0.001 Å. At much longer times (≈1 ns), when the change inexpansion levels off, a temperature of 510 K would correspond to alinear expansion of 0.0038 Å, and our experimental value is0.0032±0.0005 Å (see FIG. 18B). This temporal decrease in the disparityin spacing is indicative of the change in surface to bulk-type behavior.From modeling of strain propagation in X-ray studies, a 0.0082-Å surfacestrain amplitude was inferred for GaAs. Our measurements indicate alarger deformation (by a factor of 3).

Because of their small incidence angle, the probing electrons have avery small penetration depth (a few Å for 30-keV electrons at θ_(i)≈1°)and thus probe only the very top surface layers of the crystal; in thegeometry of our experiment, the excitation pulse (30° incidence angle)has a vertical penetration depth also of nm scale (3.5 nm at 266 nm).These small and comparable penetration depths for the photons andelectrons provide a unique condition for monitoring the local structuraldynamics of these surface layers. In X-ray experiments, a heating pulsetypically has an absorption length of 0.3 μm, and the probing X-raypulse has a micron-scale penetration depth. Such scales generallyrequire consideration of strain propagation in the bulk and over lengthson the order of microns. Clearly, direct probing of the surface motionsof atoms provides insight into the understanding of the surface initialdynamics and to the connection to bulk propagation at differenttemperatures (fluences). The effect of fluence on structural changes ismapped out in FIG. 19A, and representative examples of experimentaltraces that follow the time-dependent Bragg reflection are shown in FIG.19B.

Additional experiments were carried out on silicon to isolate the effectof chlorination and to test the generality of the approach and the scopeof application. Both chlorinated and non-chlorinated silicon (111)surfaces were subjected to the same experimental conditions (excitationwavelength and fluence). Similar behavior to that of GaAs wasfound—whereas hydrogen-terminated silicon did not present noticeablesurface contraction preceding the expansion, the chlorinated surfaceshowed a prompt contraction, which precedes the expansion. The overallphase of the signal in FIG. 18 depends on the overlap at the mask and itis possible that optical phonon generation at t=0 contributes to theobserved scattering. It should be noted that the phase corresponding tothe expansion is unequivocally established and confirmed by the temporalevolutions of the temperature and structural dynamics at differentinfluences. However, the observations with chlorine are consistent witha potential-driven change at the earliest times, prior to coherentacoustic phonon generation.

Based on these experiments, a possible mechanism is suggested.Embodiments of the present invention are not limited to this suggestedmechanism. One can argue that a general structural-dynamics picture nowemerges from the observations of the structure changes and thetimescales of the motion (see FIG. 18B), and from the observations onsilicon surfaces. In the nonthermal regime, the initial femtosecondtransient excitation, which creates the electron-hole pairs, distortsthe potential, and structural changes occur on the ultrashort timescaleby this deformation prior to significant motion in the lattice(phonons), as experimentally verified above. This highly non-equilibriumstate of the solid is followed by energy dissipation and redistribution,which ultimately lead to expansion of the lattice and restructuring atlonger times. With this in mind, only an expansion of the surface atomswould be expected, contrary to the contraction and expansion observed inthe studies reported herein. However, for the chlorine-terminatedsurface, the large electronegativity shifts the electronic chargedistribution towards the chlorine (ionic potential). The ensuingCoulombic interaction with the underlying layers contracts theinteratomic layers, as observed in the early-time ultrafast rise of thecontraction (see FIG. 18A), and on this timescale, the dynamics isdriven by the potential change. Along with the observations made in thecase of silicon and supporting this proposed mechanism for contraction,we note that atomic chlorine chemisorbed on GaAs was found to be anelectron acceptor.

Following the contraction, expansion proceeds on a similar timescale.Through Auger processes (at a density of ≈10²¹ cm⁻³), which take placein a few ps, the carrier density decreases, but the total electronicenergy remains unchanged. The drop in the Coulombic potential along withelectron-phonon coupling now drives the system in a reversed motiontoward expansion (see FIG. 18A). The expansion of the lattice typicallyrequires 7 ps to define surface-layer temperature and this is evident inthe rise of the intensity profile (see FIG. 18B); only after this risecan we define the temperature acquired through electron-phonon coupling.The structural change (expansion) follows the temperature rise, butafter the apparent delay of 15 ps, reaching its maximum of +0.025 Åexpansion at yet longer time. This thermal expansion in the (111)direction is likely due to anharmonicity of lattice vibrations.Remarkably, the width of the Bragg spot reaches its maximum before thepeak of structural lattice expansion. Lattice dynamics is first drivenby coherent collective phonons followed by the isotropic expansion thatensues when anharmonicity becomes effective. This nascent latticeexpansion generally should first overcome the persisting contraction(see FIG. 18A). From our data, we obtained an onset for the expansion of≈5 ps after the temperature has risen to half of its maximum and anadditional delay of ≈10 ps to overcome the initial contraction. Itshould be noted that this picture of structural dynamics is robust atlower fluences as demonstrated by FIG. 19. However, in the lower fluenceregime, the initial temperature is decreased, electron-phonon couplingdominates, and diffusive processes become pronounced at longer times.

The return to the original structure is observed in the decrease inΔd₁₁₁, from +0.025 to +0.003 Å, but this restructuring takes place on amuch longer timescale (see FIG. 18). We note that diffusive processestypically begins beyond 50 ps, as up to this time Δd₁₁₁ continues toincrease—cooling down the surface by diffusion leads to a decrease inΔd₁₁₁. Thus, the structure at the expanded value of Δd₁₁₁=0.025 Å isvibrationally in a non-equilibrium state of collective modes, whichcools down by energy redistribution and diffusion at longer times.Theoretical calculations of the heat diffusion using the known thermalproperties of GaAs (heat capacity and thermal conductivity) provide agood match to the temperature behavior from the point of leveling offshown in FIG. 18B.

Thus, this experiment demonstrates that new dimensions of ultrafastelectron crystallography at time, length, and sensitivity scales areideally suited for atomic-scale structural dynamics of surfaces andinterfaces. One reason this is achieved is because of the ability todirectly determine—from the diffraction periodicity and changes withtime—the evolution of atomic spacings (Bragg peak positions), transienttemperatures (Bragg integrated intensity), and the involvement ofcoherent lattice vibrations (Bragg peak width). The timescales for thenon-equilibrium surface structures, and restructuring at longer times,are desirable for understanding the surface phenomena and forpropagation in bulk materials. We believe that embodiments of thepresent invention now provide for the advancement of many applicationsin this general area of surface science and nanometer-scale materials,and macromolecular structures.

In other experiments using a system according to one embodiment of thepresent invention, the temporal evolution of bulk and surface structureswith atomic-scale spatial resolution was studied. Merely by way ofexample, one experiment, crystals of silicon, with and withoutadsorbates, were studied. FIG. 20 illustrates results obtained duringstudies of condensed phases, e.g. crystals and solid-liquid phasetransitions. In FIG. 20A, diffraction from the Si crystal isillustrated. In FIG. 20B, diffraction frame referencing of Bragg spotsis shown. FIG. 20C illustrates diffraction frame referencing ofamorphous-to-liquid transition.

Referencing the diffraction difference as illustrated in FIG. 20 to theground-state shows the changes in the structure caused by the initiatingpulse, from ground-state pattern at negative time to the observed changeat positive time. The structural change is evident in the shift withtime of the in-phase Bragg peak of the rocking curve, while the increasein vibrational amplitude is reflected in the broadening. The changetakes place as a rise to a maximum shift and then a decay to thecoordinates of the original structure. By gating on a Bragg spot, we canfollow the changes with time. These results show the “instantaneous”structural change (2 ps steps of change; total of 10 ps); a homogeneousexpansion of the lattice 2.35 Å Si—Si bond by up to 0.04 Å at 50% levelof damaging fluence. This is followed by lattice relaxation from ahighly non-equilibrium structure to the final ground-state structurewith Si—Si distances of 2.35 Å. The multiple time constants ofrestructuring describe the expansion in real space and vibrationaltemperature.

Additional studies for the surface structure (and with hydrogen orchlorine), produced striking results. Gating the streak at theout-of-phase condition, embodiments of the present invention enable thesurface structural interferences and their evolution with time to bedirectly resolved. Remarkably, the two spots change in time, anddifferently, but maintain the phase coherence. We are therefore able todisplay the spatial, temporal and phase coherence change of the surfacestructure, and the spatial patterns are describable within the frameworkof diffraction (kinematic) theory of condensed matter. The change in thestructure is evident in the amplitude change of surface Si atoms. Unlikethe result for bulk displacement (0.04 Å), the expansion of surfaceatoms is larger by an order of magnitude (0.4 Å). The cooling of thesurface structure occurs on a time-scale different from that of thebulk. We also studied the structural changes involved in phasetransitions when the temperature of the lattice is sufficiently high tocause large amplitude disorder. Initiating an ultrashort temperaturejump of the amorphous structure with the infrared femtosecond pulsegives new diffraction ring patterns, which we followed as a function oftime by referencing to the image of the ground state (see FIG. 20). Thestructural change is a phase transition to the liquid state.

The examples and embodiments described herein are for illustrativepurposes only. Various modifications or changes in light thereof will besuggested to persons skilled in the art and are to be included withinthe spirit and purview of this application and scope of the appendedclaims. It is not intended that the invention be limited, except asindicated by the appended claims.

1. A system for imaging one or more samples, the system comprising: astage assembly adapted to receive a sample to be imaged; a laser source,the laser source being capable of emitting an optical pulse; a cathodecoupled to the laser source, the cathode being capable of emitting anelectron pulse having about one to 1000 electrons; an electron lensassembly adapted to focus the electron pulse onto the sample disposed onthe stage; a detector adapted to capture one or more electrons passingthrough the sample, the one or more electrons passing through the samplebeing representative of an image of the sample, the detector providing adata signal associated with the one or more electrons passing throughthe sample that represents the image of the sample; a processor coupledto the detector, the processor being adapted to process the data signalassociated with the one or more electrons passing through the sample tooutput information associated with the image represented by the sample;and an output device coupled to the processor, the output device beingadapted to output the information associated with the image representedby the sample.
 2. The system of claim 1 wherein the laser sourcecomprises a mode-locked laser oscillator providing a train of opticalpulses with a Full Width Half Maximum (“FWHM”) of less than 500 fs. 3.The system of claim 2 wherein the FWHM is less than 100 fs.
 4. Thesystem of claim 1 wherein the cathode comprises at least one of a LaB₆bearing crystal, a ZrC bearing crystal, or a CeB₆ bearing crystal. 5.The system of claim 1 wherein the detector comprises a digital chargecoupled device (“CCD”) camera.
 6. The system of claim 1 wherein theprocessor comprises an image processor associated with a transmissionelectron microscope (“TEM”).
 7. The system of claim 1 wherein the sampleis selected from a biological sample, a chemical sample, a physicalsample, or an electronic sample.
 8. The system of claim 1 wherein theoptical pulse is one of a plurality of optical pulses.
 9. The system ofclaim 1 wherein the sample is maintained in a vacuum.
 10. The system ofclaim 1 wherein the sample is maintained at a temperature ranging fromabout 77 K to about 300 K.
 11. The system of claim 1 wherein the systemfurther comprises one or memories including computer code, the computercode being adapted to control at least one of the electron lensassembly, the detector, the processor, or the output device.
 12. Atransmission electron microscope (TEM) for acquiring time-resolvedimages of a sample, the TEM system comprising: a stage assembly adaptedto receive a sample having a feature to be imaged, the feature having asize; a vacuum chamber enclosing the stage assembly; a laser sourceadapted to provide a photon pulse; a cathode coupled to the lasersource, the cathode being capable of: emitting one or more first pulsesof electrons toward the feature of the sample, the one or more firstpulses of electrons each having about one to about 1000 electrons; andemitting one or more second pulses of electrons toward the feature ofthe sample, the one or more second pulses of electrons each having aboutone to about 1000 electrons; a detector adapted to: capture a firstportion of the one or more first pulses of electrons device during afirst portion of time, the portion of the one or more first pulses ofthe electrons being associated with a first image of the feature of thesample during the first portion of time; and capture a second portion ofthe one or more second pulses of electrons using the sensing deviceduring a second portion of time, the portion of the one or more secondpulses of electrons being associated with a second image of the featureof the sample during the second portion of time; and a processor adaptedto: receive first information associated with the first portion of theone or more first pulses of electrons during the first portion of timefrom the detector; and receive second information associated with thesecond portion of the one or more second pulses of electrons associatedduring the second portion of time from the detector.
 13. The TEM systemof claim 12 wherein the one or more first pulses of electrons each hasan average temporal pulse width of less than 1 ps.
 14. The TEM system ofclaim 13 wherein the one or more second pulses of electrons each has anaverage temporal pulse width of less than 1 ps.
 15. The TEM system ofclaim 12 wherein the one or more first pulses of electrons each hasabout one to about 1000 electrons and the one or more second pulses ofelectrons each has about one to about 1000 electrons.
 16. The TEM systemof claim 12 wherein the size is less than 100 nanometers.
 17. The TEMsystem of claim 12 wherein the first image has one or more differentcharacteristics from the second image.
 18. The TEM system of claim 12further comprising a beam splitter adapted to direct a portion of thephoton pulse to the feature of the sample during the first portion oftime and not during the second portion of time.
 19. The method of claim12 further comprising a beam splitter adapted to direct a portion of thephoton pulse to the feature of the sample during the second portion oftime and not during the first portion of time.
 20. The method of claim12 wherein the first portion of time is on an order of seconds and thesecond portion of time is on an order of seconds.